2 MATERIALS AND METHODS
2. 1 Clinical data
For the purposes of this study, 42 consecutive participants were prospectively recruited: 24 (7 female, 17 male; Group A ) had leukoplakia and 18 (4 females and 14 males) had glottic cancer (pT1, n=6; p T2, n=8; pT3, n=8; Group B ). Regarding the patients with malignancies, 8 had bilateral lesions and thus 26 malignant lesions were totally detected by narrow band imaging. Between patients with non-malignant lesions ,8 of them had bilateral lesions of the vocal cords, therefore we have examined a total of 32 nonmalignant lesions. Regarding basic characteristics, there was no difference in age, number of cords affected, cigarettes smoked per year and years smoking between male and female patients (Table 1 ). For the purposes of our study we have also examined 42 non-smokers (Control Group-Group C ), who received total anesthesia for surgeries, such as hernias, colectomies etc, which had nothing to do with otolaryngological diseases..
2.2 Contact endoscopy
For contact endoscopy the Andrea-Dias Contact Micro Laryngoscope (with HOPKINS Straight Forward Telescope 0° and 30°, with diameter 5.5 mm, length 23 cm, magnification 60 × and 150×); a 3 chip camera (Tricam SL II); a Xenon 175 watt light source and a video recording system (AIDA) were used, all manufactured by Karl Storz, (Tuttlingen, Germany).
The entire larynx was initially visualized with standard white light, followed by visualisation using the narrow band imaging (NBI) mode. Endoscopically guided biopsy of laryngeal lesions was also performed; tissue was fixed in 10% formalin for histological analysis.10,11 The recorded findings were examined by two persons (PP and VST), who evaluated separately the pictures before discussing together the results. All of them were blinded to the histological results. The interrater reliability was also calculated with the use of Kappa test was estimated at 0.89 (Cohen’s kappa statistic).
2.3 Morphological types of the surface of the vocal cords
The morphological types of vocal fold leukoplakia assessed by preoperative rigid laryngoscopy were categorized as: flat and smooth, elevated and smooth, and rough type.11
The definition is presented as the following:
Flat and smooth type: Surface: smooth; Margin: lesion without raised margins, being continuous with the surrounding mucosa; Texture: homogeneous, regular, the lesion with even coloration.
Elevated and smooth type: Surface: smooth; Margin: lesion with raised margins, sharply demarcated from the surrounding mucosa; Texture: homogeneous, regular, the lesion with even coloration.
Rough type: Surface: wrinkled, corrugated; Margin: lesion with raised margins, sharply demarcated from the surrounding mucosa; Texture: non-homogeneous, irregular, the lesion with uneven coloration and is usually accompanied with erosion or ulceration.
2.4 Patterns and changes
The Ni categorization was used for the purposes of our research.12 Intraepithelial capillary loop alterations seen with Contact Endoscopy (CE) can be categorized into five categories (I to V) according to this classification. Intraepithelial papillary capillary loops are nearly inconspicuous in type I, while oblique and arborescent capillaries of small diameter are discernible. The intraepithelial papillary capillary loops are nearly invisible in type II, while the diameter of the clearly apparent oblique and arborescent capillaries is increased. The mucosa is white in type III, and the intraepithelial papillary capillary loops are invisible; if the white patch is thin, the oblique and arborescent vessels can be seen indistinctly, but if the white patch is thick, the vessels are obscured. The mucosal intraepithelial papillary capillary loops appear as scattered, small, dark brown spots in type IV, with a relatively regular arrangement and low density; the capillary terminals are bifurcated or slightly dilated, and the intraepithelial papillary capillary loops appear as scattered, small, dark brown spots; the oblique and arborescent vessels are usually not visible.12
Type V changes are subdivided into types Va, Vb andVc according to the shape, regularity and distribution of vessels. In type Va, intraepithelial papillary capillary loops are significantly dilated and of relatively high density, and appear to be solid or to have hollow, brownish, speckled features and various shapes.12 In type Vb, the intraepithelial papillary capillary loop itself is destroyed, with its remnants presenting in a snake-, earthworm-, tadpole- or branch-like shape, and the microvessels are dilated, elongated and ‘woven’ in appearance. In type Vc, the lesion surface is covered with necrotic tissue, and the intraepithelial papillary capillary loops present as brownish speckles or tortuous shapes of uneven density which are irregularly scattered on the tumor surface.12
According on the shape, regularity, and distribution of vessels, type V changes are split into types Va, Vb, and Vc. Intraepithelial papillary capillary loops in type Va are highly dilated and of relatively high density, appearing solid or hollow, brownish, speckled, and of varied shapes.12 The intraepithelial papillary capillary loop is disrupted in type Vb, with remains resembling a snake, earthworm, tadpole, or branch, and microvessels that are dilated, elongated, and ’woven’ in appearance. The lesion surface is coated with necrotic tissue in type Vc, and the intraepithelial papillary capillary loops appear as brownish speckles or sinuous shapes of uneven density spread irregularly on the tumor surface.12,13 Type is depicted inImages 1, 2, and 3 .
2.5 Histologic examination
All the tissues were processed for pathological testing on a regular basis. The same pathologist evaluated and graded histologically graded formalin-fixed and paraffin-embedded slides independently. Squamous cell hyperplasia with non-dysplasia, mild dysplasia, moderate dysplasia, severe dysplasia, carcinoma in situ, and squamous cell carcinoma were all assessed histologically according to the World Health Organization’s 2017 guidelines.14 The new WHO 2017 classification is a two-tier system. Laryngeal precursor lesions are classified as low-grade dysplasia (previous categories squamous hyperplasia, mild dysplasia), and high-grade dysplasia (previous categories of moderate and severe dysplasia, carcinoma in situ).14 Carcinoma in situ, is distinguished from high-grade dysplasia, showing features of conventional carcinoma.14
2.6 Statistical analysis
Parameters were evaluated using the Jamovi project (2021; Jamovi, software Version 1.6, Sydney, Australia. Retrieved from www.jamovi.org). A p-value less than 0.05 was considered statistically significant for all analyses. Independent samples t-test, Mann-Whitney U test and Chi square test were used for basic characteristics’ comparisons between male and female patients’ features (age, years of smoking, number of cigarettes/day) as well as for comparisons between patients with unilateral or bilateral lesions and patients with or without histologically confirmed malignancies. The ANOVA and the nonparametric Kruskal-Wallis tests were used to detect possible statistically significant differences between lesions with different vascular patterns.