2. 1 Clinical epidemiologic data
Seventy-nine patients (21 female, 58 male), with a mean age of 57.5 years ± 7.12 (range, 32–73 years), were prospectively enrolled and evaluated.
Of these patients, 58 had leukoplakia (Group A), and 21 had malignant lesions (pT1, n=6; p T2, n=8; pT3, n=8; Group B), as proven by the results of the histological examination. Regarding the patients with malignancies, 8 had bilateral lesions, and thus 29 SCC lesions were studied and detected with CE. Between patients with leukoplakia and no malignancy, there was a total of 77 lesions. Therefore, a total of 106 lesions (29 malignancies/squamous cell carcinoma and 77 non-SCC) were found. Concerning the leukoplakia-patients, by 29 of them a low-grade dysplacia has been observed, and the rest of them suffered from a high-grade one. By the tumor-suffering patients, all of them had a high grade SCC.
In Table 2, demographic features, such as smoking status and age, of patients with and without SCC are depicted and compared.
For this study, we have additionally examined 79 non-smokers (control group-group C), who received total anesthesia for surgeries for non-otolaryngological diseases, such as hernias or other abdominal surgeries.
The study protocol was reviewed and approved by the Institutional Review Board ( Papanikolaou General Hospital, Thessaloniki, Nr. 72-2204202). All participants provided informed consent for participation in the study after being instructed about the aim and the procedures of the study. The procedures were in accordance with local data protection guidelines and legislation and according to the principles of the Helsinki Declaration for studies in humans.
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 have been used, all manufactured by Karl Storz (Tuttlingen, Germany).
Vascular patterns were studied before staining because the staining dye causes a loss of transparency of the vocal cord mucosa making the blood vessels invisible. Epithelial cellular architecture was studied after staining the mucosal surface with 1% methylene blue which imparts a dark blue color to the nucleus and a light blue color to the cytoplasm. The excess stain has been removed by washing the area with a copious amount of saline using suction and irrigation. Methylene blue is non-toxic and the staining is reversible [8.9.11].
Endoscopically guided biopsy of laryngeal lesions was also performed under general anesthesia; tissue specimens were fixed in 10% formalin for histological analysis [10,11]. The recorded CE findings were examined by two experienced professionals (PP and VST), who evaluated the pictures separately before discussing the results together. Both of them were blinded to the histological results at the time of assessment of the CE findings. The interrater reliability was calculated with the use of the Kappa test and was equal to 0.89 (Cohen’s kappa statistic).
2.3 Morphological types of the surface of the vocal cords
The morphological types of vocal cord leukoplakia assessed by preoperative rigid laryngoscopy were categorized as flat and smooth, elevated and smooth, and rough type [11,12].
These definitions were based on the following morphological pattern categories, as depicted in Table 1.
2.4 Patterns and changes
The Ni categorization was used for our research.16 Intraepithelial capillary loop alterations seen on 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 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 [13].
Type V changes are subdivided into types Va, Vb, and Vc 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 [13]. 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 [13].
According to 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.16 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 [12-14]. The various type categories are depicted in Figures 1, 2, and 3.
2.5 Histologic examination
All the tissues were processed for pathological testing on a standard basis. The same pathologist, who was blinded to the CE findings, 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 (WHO) 2017 guidelines [15]. The 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). Carcinoma in situ is distinguished from high-grade dysplasia, showing features of conventional carcinoma [15].
2.6 Statistical analysis
Parameters were evaluated using jamovi software (Version 1.6, Sydney, Australia 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 association between the appearance of the surface of the vocal cords and the vascular patterns was examined in both groups of leukoplakia and glottic cancer, as well as in healthy vocal cords using the Chi-square test or Fisher’s exact test (if expected counts were less than 5). The association between the healthy cord’s vascularization pattern and the presence or absence of malignancy in the contralateral cord was investigated using the Chi-square test or Fisher’s exact test.