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.