Abstract
In India cancer patients come at a very advanced stage with many of them being crossed the stage of resection. A wide range of non invasive techniques like toludine blue staining, methylene blue staining, Narrow band imaging have been developed for the early detection of malignant and premalignant lesions in the mucosa including oral cavity and gastrointestinal tract. However it is difficult for the clinicians to decide which diagnostic tool is most appropriate and useful for screening, resulting in failure to pick up the lesions at an early stage. Various online journals have been reviewed and no journal was found to support this point. This study here by describes Toludine blue and narrow-band imaging (NBI), diagnostic tools already proven independently as a useful screening method in many fields, and demonstrate its usefulness in the early detection of premalignant and malignant lesions of the oral cavity, as reported by previous studies in the otolaryngologic literature and compare both screening tools which has not been done till now. This study was done in a tertiary referral centre in middle India from March 2018 to August 2019 in order to evaluate the role of different screening tools (NBI and Toludine blue).44 patients with suspicious oral cavity lesions (premalignant and malignant) who had given consent for both NBI and toludine blue screening techniques were selected from the suspected (premalignant and malignant lesions) who had visited the OPD during the study period. Patients with proven malignancy were excluded from the study. A detailed history of the patient taken and standard Ear, nose, throat, oral cavity and neck examination of patient carried out. After subjecting the patients to screening tools like NBI and toludine blue, the efficacy of these techniques in helping us to take a representative biopsy was evaluated. In present study the sensitivity (to correctly identify all patients with disease), specificity (to correctly reject healthy patients without disease) of older technique toludine blue in detecting premalignant lesion was 66.6%, 87.8% while for NBI was 66.6%, 95%. For malignant lesion sensitivity and specificity of toludine blue was 94.3%, 100%, while the same for NBI was 100%, 88.8% respectively. Both NBI and toludine blue staining can be adopted for screening and the accurate detection of biopsy site and in the follow up of premalignant lesions to look for malignant transformation. Time tested is Toludine blue which is cheap and easily available. But being a better tool and having an upper hand in evaluating the lesions, NBI should be made available in all the secondary and tertiary care centres as a screening method.
Keywords: DNA-deoxyribo nucleic acid, RNA-ribonucleic acid, NBI-narrow band imaging, NBI ME-narrow band imaging magnification endoscopy, WHO-World Health Organisation, OSCC-oral squamous cell carcinoma, TB-toludine blue, PPV-positive predictive value, NPV-negative predictive value
Introduction
Head and neck cancer is one of the most common cancers in our country among which oral cavity cancer is the major one. Over 200,000 cases of head and neck cancer occur each year in India among them 80,000 oral cancers are diagnosed each year in our country [1]. The development of oral cancer is thought to occur in a step wise fashion. A precancerous lesion is a morphologically altered tissue in which cancer is more likely to occur than its normal counterpart. The cellular changes were referred as atypia and the general disturbance in epithelium as dysplasia. The cellular characteristics are what define the risk of progression and these lesions can demonstrate mild, moderate, severe dysplasia, which can then evolve to carcinoma in situ and ultimately to invasive cancer.
An early lesion of oral cavity may be missed due to their close resemblance to benign/premalignant lesion. The biggest challenge in the diagnosis lies in deciding the site of biopsy in early lesions and sometimes whether or not a biopsy is required in these lesions. Thus identifying clinically suspicious/undetectable lesions has gained importance whereby diagnosis can be confirmed by biopsy of representative site at an earlier stage.
A wide range of non invasive techniques have been developed for the early detection of malignant and premalignant lesions. Among which Toludine blue was the oldest one lasting for more than 50 years. Toluidine blue is an acidophilic meta chromatic dye which selectively stains acidic tissue components, thus staining DNA and RNA. As it binds to nucleic acids (DNA or RNA), it helps in better visualization of high risk areas especially with rapid cell proliferation of SCC and premalignant lesions It was used not only in oral cavity but also in cervical cancer screening and it has stood the test of time.
NBI is an advanced imaging system which uses optical image enhancing technology to see endoscopic images and to improve the visualisation of mucosal architecture and microvascular pattern. It increases tissue contrast by specifically identifying superficial capillaries and neo-angiogenesis in abnormal mucosa. Lesions which showed irregular borders with weaving, irregular and increase vascularity with increased capillary loops (IPCLs-brown dots) were considered as malignant.
Muto et al. in 2004 [2] were the first to use a prototype NBI system for screening the oropharynx and hypopharynx for SCC in patients with oesophageal cancer. It requires no special dyes and allows for easy inspection of the superficial vascular bed. This study here by describe Toludine blue and narrow-band imaging (NBI), diagnostic tools already proven as a useful screening method in many fields, and demonstrate its usefulness in the early detection of premalignant and malignant lesions of the oral cavity, as reported by previous studies in the Otolaryngologic literature and compare both screening tools which has not been done till now Early histopathological confirmation not only helps in initiating early treatment but also improves the compliance and follow up of the patient. The aims and objective of this study is
To evaluate the application of NBI and Toludine blue in the early screening of premalignant and malignant oral cavity lesions.
To compare the benefits of N.B.I and Toludine blue and add on benefit in doing both in the same patient.
To evaluate the advantages, clinical applicability and the feasibility of above mentioned screening tools in the current scenario which has not been done till now.
Materials and Methods
This prospective clinical study was carried out in the Department of Otorhinolaryngology and Head and Neck Surgery of a tertiary referral centre in middle India from March 2018 to August 2019. 44 patients with the lesions in oral cavity having suspicion of malignancy were clinically selected. Patients with proven malignancy were not included in the study. After taking the detailed history and clinical examination, patients underwent zero degree endoscopic examination in White Light, followed by NBI. After examining these lesions, photograph of the suspected area was taken. Then the patients with slight positivity like lightly or darkly stained with Toludine blue and altered vascularity with NBI were processed for biopsy. Punch biopsy was taken from the site after applying topical anaesthetic agent. If both showed the same area, single biopsy was taken, otherwise biopsy from 2 different sites taken and were send for histo -pathological examination in formaline vial. Patients were followed up with the HPR report.
Observations and Results
Total 44 patients qualified to be included in the study, in which both screening methods were administered. In these patients the most common presenting complaint was non healing ulcer in oral cavity. Most common site of lesion was found to be buccal mucosa followed by tongue, gingivobuccal sulcus, alveolus, floor of mouth, retromolar trigone and lip. These patients were subjected to toludine blue staining and NBI.
Results of Toludine Blue Staining
Toluidine blue dye is an acidophilic dye that selectively stains acidic tissue components such as DNA and RNA. Since dysplasia and in situ carcinoma contain much more DNA and RNA than the normal surrounding epithelium, these lesions preferably takes up toludine blue.So the lesions that showed dark blue staining were considered to be positive for premalignant or malignant tissue, while those with light staining, or totally not coloured, were considered negative. Out of 44 cases, 33 were darkly stained (indicative of malignancy), 7 were light stained (non malignant/inconclusive), 4 didn’t take stain at all (suggestive of non malignant lesion) (Fig. 1).
Fig. 1.
Results of toludine blue staining
Correlation of Staining with H.P.R
Out of the 33 cases which took up the stain (dark blue) by toludine blue method all were found to be malignant as per the H.P.R report. Out of 11 cases which did not stained/light stained, 6 cases were found to be benign, 3 were precancerous and 2 were malignant. Thus Toludine blue picked up 33 malignant lesions where as missed 2 malignant cases as well as 3 precancerous lesions which is a significant error (Fig. 2).
Fig. 2.
Correlation of staining with H.P.R
Results of Narrow Band Imaging
On NBI altered or hyper vascularity and increased capillaries (brown dots) suggestive of malignancy, while uniform or normal vascularity rules out the malignant chances.
Out of 44 cases, 36 shows altered vascularity (significant)where as 8 showed normal vascularity(non significant) (Fig. 3).
Fig. 3.
Results of narrow band imaging
Correlation of Narrow Band Imaging with H.P.R
Out of the 36 altered vascular cases by NBI, 35 were found to be malignant and 1 was precancerous and from the 8 normal vascularity lesions shown by N.B.I, 6 were benign and 2 were found to be precancerous. Thus NBI picked up all the malignant cases and missed 2 precancerous lesions (Fig. 4).
Fig. 4.
Correlation of narrow band imaging with H.P.R
Overall Diagnostic Validity of NBI and Toludine Blue
By Comparing Toludine Blue Staining and N.B.I Endoscopic Findings with Their H.P.R Reports
Out of the 33 darkly stained lesions by toludine blue, 33 were histologically proved malignant, and out of 11 lightly stained lesions 6 were benign, 3 premalignant and 2 malignant lesions. In narrow band imaging, from the 36 altered vascularity lesions 35 were histologically proved malignant and 1 was premalignant where as from the 8 normal vascularity lesions 6 were benign and 2 were premalignant (Fig. 5).
Fig. 5.
Overall diagnostic accuracy of NBI and toludine blue
Statistical Analysis of Toludine Blue Staining and Narrow Band Imaging
The sensitivity, specificity, PPV, NPV of toludine blue in detecting premalignant and malignant lesions were 66.6%, 87.8%, 28.5%, 97.2% and 94.3%, 100%, 100%, 81.8% respectively while the same for narrow band imaging was 66.6%, 95%, 50%, 97.5% and 100%, 88.8%, 97.2%, 100% respectively (Fig. 6).
Fig. 6.
Statistical analysis of toludine blue staining and narrow band imaging
Discussion
Since dysplasia and in situ carcinoma contain much more DNA and RNA than the normal surrounding epithelium, the use of in vivo staining, by means of toluidine blue dye, is based on the fact that it is an acidophilic dye that selectively stains acidic tissue components such as DNA and RNA. After the thorough clinical examination, the suspicious lesions were stained with Toludine blue. The biopsy site was selected on the basis of dye retention. Lesions that showed dark blue staining were considered to be positive for premalignant/malignant tissue, while those with light staining, or totally not coloured, were considered negative (Fig. 7).
Fig. 7.
Staining of malignant lesion by toludine blue. Toludine blue also stains normal taste bud
In the present study, sensitivity of toludine blue in detecting premalignant or malignant lesion was 94.3% and the results were in accordance with Shambulingappa Pallagatti who found the sensitivity to be 95%. The sensitivity was 94.3% because Toludine blue only lightly stained 2 lesions which histopathologically came out as squamous cell ca. The results of sensitivity of toludine blue differed from the findings of Juhi Upadhyay, Kamarthi Nagaraju et al., Mahesh Chandra hedge which were 73.9%, 100%, 97.29% respectively. The difference in sensitivity may be attributed to difference in methodology of staining (Table 1).
Table 1.
Stastical analysis of various toludine blue studies till date
| Study | No of pts. | Sensitivity (%) | Specificity (%) | False positive | False negative | PPV | NPV |
|---|---|---|---|---|---|---|---|
| Present study | 44 | 94.30 | 100 | O | 57.10% | 100% | 81.80% |
| Pallagatti et al. [3] | 37 | 95 | 71.45 | 84.60% | 90.90% | ||
| Upadhyay et al. [4] | 47 | 73.90 | 30 | 32.60% | 26.10% | 54.80% | 50% |
| Nagaraju et al. [5] | 60 | 100 | 100 | 100% | 100% | ||
| Hedge et al. [6] | 90 | 97.29 | 62.50 | ||||
| Onofre et al. [7] | 50 | 77 | 67 | 43.50% | 88.90% | ||
| Joel Bepstein et al. [8] | 59 | 92.50 | 63.20 | ||||
| Waruakulasuriya et al. [9] | 145 | 100 | 62 | 20.50% | |||
| Mashberg [10] | 235 | 98 | 92 | 8.50% | 6.70% | ||
| Vahidy et al. [11] | 1190 | 86 | 80 | ||||
| Neibel and Chomet [12] | 20 | 100 | 100 |
In our study the overall specificity of TB was found to be 100%, which was in concordance with Kamarthi Nagaraju et al. and Neibel HH, Chomet B. It was different from the results by Shambulingappa Pallagatti, Juhi Upadhyay. It may be due to variation in patient selection (Table 1).
In the present study, the PPV, NPV, of TB was 100 and 81.8% respectively. The findings had similarity to study by Kamarthi Nagaraju et al. which was 100% both. The fall in our NPV is because 2 of our malignant lesions failed to retain the stain. The study by Onofre Ma had PPV of 43.5 and NPV of 88.9% respectively. The fall in PPV is due to faulty uptake of stain by benign lesions.
Principle of Narrow Band Imaging (NBI)
NBI is a non-invasive technique for visualization of oral mucosal vascular structure. It relies on the principle of depth of light penetration and the absorption peak of haemoglobin. NBI system contains a special image processor and a lighting unit with special filters that narrow frequency range of white light to 400–430 nm (centred at 415 nm, i.e., blue) and 525–555 nm (centred at 540 nm, i.e., green) bands. 415 nm wavelength (blue) light has less penetration and less scattering, thus enhancing image resolution. The blue filter is designed to correspond to the peak absorption spectrum of haemoglobin to enhance the image of capillary vessels (intraepithelial papillary capillary loops [IPCL]) on mucosal surface. 540 nm (green) wavelength light penetrates deeper and highlights the submucosal vascular plexus. Capillaries in the superficial mucosal layer are displayed in brown, whereas deeper mucosal and submucosal vessels are displayed in cyan. The reflection is captured by a CCD chip and an image processor creates a composite pseudocolor image, which is displayed on a monitor, enabling NBI to enhance mucosal contrast.
Kamath et al. defined patterns of narrow band imaging [13].
In normal mucosa of oral cavity Superficial epithelial layer and blood vessels appear as brown (penetrated by 415 nm (blue) light and Sub epithelial layer appeared as cyan or green (penetrated by 540 nm green) light. Normal mucosal vasculature appeared as clear branching vessels.
Neoplastic lesions appeared as areas with scattered spots with a well demarcated border and superficial vessels of these lesion appeared as intra-papillary capillary loops (IPCL) which were represented by brown spot Abnormal vasculature within sub epithelial layer was shown as brownish dots with extension, dilatation, weaving and differing shapes. The Usual branching patterns of normal capillary beds is lost. The extent of lesion can be better identified by identifying the area of vasculature, which will help in identifying site of biopsy and resection margins. Alteration in degree of dilation, meandering and tortuosity of IPCLSs represent severity of dysplastic/malignant pathology.
This pattern has been followed in my studies to analyse the lesions (Fig. 8, Table 2).
Fig. 8.
a Oral mass examined under white light endoscope b NBI endoscopy Ill defined lesion with altered vascularity and weaving pattern suggestive of malignancy. Histopathology report confirmed the diagnosis
Table 2.
Statistical analysis of various nbi studies till date
In my present study the sensitivity of NBI in detecting the malignant lesion was 100% while specificity was 88.80%. NBI showed altered vascularity with brown dots in 2 lesions which came out to be precancerous. The specificity in my study may be less compared to other studies may be due to various reasons. When it comes to the field of otolaryngology and head and neck surgery, the literature on the use of NBI is very sparse. So there are only few studies to compare. Second my study population may be small. Variation can also be due to the change in the diagnostic criteria. In some studies, they regarded a well-demarcated brownish area with scattered brown spots as susceptible lesions. Some studies used Ni’s classification, the classification of the microvascular endoscopic patterns of Ni et al. [18] Additionally, some studies used visualize intrapapillary capillary loops (IPCLs) pattern and a color change in the area between IPCLs. In my study I followed Kamath et al. classification [13]. Uneven standardization greatly reduced the generalizability and resulted in variation of opinion in experts in interpreting the results. However the time of detecting the lesion, the mucosal lesion size, the sample size, the pathological type and the bias of selection of patients can also reduce the generalizability of the overall performance and increase the heterogeneity of the various studies.
Conclusion
The quick, inexpensive and non invasive method of invivo staining satisfied the requisite of detecting the suspicious lesions and an appropriate tissue specimen for histopathological evaluation for a long time. Further studies analysing a combination of sound clinical judgement and diagnostic adjuncts could be a more representative of cellular proliferations. This knowledge paved the way for invention of more and more noninvasive techniques like NBI. In my study NBI had a hand over Toludine blue in detecting the premalignant and malignant lesions of oral cavity with better sensitivity and specificity except TB having better specificity for malignant lesion. Both screening tools are having pros and cons. Toludine blue is very useful in the developing countries like India because of simple technique, cheap, cost effectiveness and is easily available, it has stood the test of time.
The advantages of NBI over toludine blue dye is that NBI is not cumbersome and is easy to operate with just the switch of a button on the videoendoscope. The diagnostic value can be enhanced by combining it with magnifying endoscopy (NBI-ME) that allows the endoscopist to zoom on the mucosa. It is a non invasive procedure. No special dyes are needed and it helps in easy inspection of lesion and superficial vascular bed. By differentiating superficial capillaries and neo-angiogenesis in abnormal mucosa, NBI enhances tissue contrast. Both NBI and toludine blue staining can be adopted for the accurate detection of biopsy site on O.P.D basis and in the follow up of premalignant lesions, with more sensitivity and least false negative value by NBI. So being a better tool and having a hand over toludine blue, Narrow band imaging should be made available in all district hospitals as a screening modality for suspicious oral cavity mucosal lesions. Thereby malignant lesions can be treated earlier, and premalignant lesions can be kept on regular follow up for 15 days, 1 month and 3 months based on the type of lesion. Earlier demarcation of malignant lesions helps to put forward an effective interventional method for the disease which reduces the mortality and morbidity of patient to a very great extent as well as it helps to curtail the functional and structural disability by disease progression.
Abbreviations
- DNA
Deoxyribo nucleic acid
- RNA
Ribonucleic acid
- NBI
Narrow band imaging
- NBI ME
Narrow band imaging magnification endoscopy
- WHO
World Health Organisation
- OSCC
Oral squamous cell carcinoma
- NBI
Narrow band imaging
- TB
Toludine blue
- PPV
Positive predictive value
- NPV
Negative predictive value
- IPCL
Intrapapillary capillary loops
- H.P.R
Histopathology report
Footnotes
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