Laryngeal cancer is one of the most common cancers of the head and neck. Early screening and diagnosis are important for maximizing the possibility of a complete cure and to preserve the voice of the patients (1, 2). Epithelial microvascular changes could be the first sign of malignant transformation of a laryngeal lesion. Using advanced endoscopic methods — narrow band imaging (NBI) or the Storz Professional Image Enhancement System (IMAGE1 S) — and on the basis of the appearance of intraepithelial papillary capillary loops (IPCLs) in the laryngeal mucosa, we can evaluate the character of these epithelial changes and perform an “optical” biopsy (3, 4). Not only visualization, but correct interpretation and evaluation of these IPCLs is crucial for early diagnosis of precancerous changes or carcinoma.

Nowadays, various descriptive classifications of microvascular changes exist (5–8). Many scientific articles discuss the sensitivity and specificity of advanced endoscopic methods in the diagnosis of dysplastic or cancer lesions using these classifications (9–18). But there is still no direct correlation between individual classification.

The evaluation of clinically and endoscopically evident benign or malignant lesions using these classifications is not complicated. However, a borderline pattern of vascularization presents scattered, small, dot-like IPCLs ( Figure 1 ). Ni et al. in their original study described this pattern in 19 patients who were histologically mostly diagnosed with hyperplasia and mild dysplasia (5). Therefore, the authors considered these IPCLs classified as type IV vasscularisation to be a benign type of vascular loops. However, the number of patients enrolled was low. On the other hand, the European Laryngological Society classification published by Arens et al. describes dot-like changes as perpendicular vascularization with malignant potential (7). Based on this discrepancy represent dot-like IPCLs, IPCLs of type IV according to Ni et al. (5), a “gray” area of interpretation of vascular changes.

The aim of this study is to clarify the clinical significance of the Ni type IV pattern of vascularization in a large group of patients.

This retrospective study enrolled patients examined and operated on between June 2015 and December 2022 at the Department of Otorhinolaryngology and Head and Neck Surgery of University Hospital in Ostrava. Data were collected in accordance with the Declaration of Helsinki, the requirements of good clinical practice, and all applicable regulatory requirements, and were approved by the Institutional Review Board.

All clinical records of the study population, including demographic features and information on previous treatments including laryngeal surgery, head and neck radiotherapy, or other treatments before the index transoral surgical procedure were retrieved from the hospital databases.

All recruited patients had symptoms of laryngeal disease  especially hoarseness or other voice problems. At first, outpatients were investigated for macroscopic laryngeal lesions (chronic laryngitis, leukoplakia, tumor, recurrent respiratory papillomatosis, cyst, polyp, granuloma, Reinke oedema). Enrolled patients had either a new, first-time-evaluated lesion, or a previously diagnosed chronic lesion (leukoplakia, recurrent respiratory papillomatosis, granuloma), which had changed, enlarged, and become symptomatic. Follow-up patients after laryngeal surgery and after radio- or chemoradiotherapy for cancer were also included. Included patients were without age limitation.

Patients who refused to undergo surgery with histological verification of a larygeal lesion or did not sign an informed consent to data processing were excluded from the study.

Epithelial microvascular changes seem to be the first sign of structural changes, and frequently also the first sign of malignant transformation of the laryngeal lesion. Based on the appearance of intraepithelial papillary capillary loops (IPCLs) detected by NBI or IMAGE1 S, we can predict the character of epithelial changes and improve detection of laryngeal carcinoma, especially in early stages of the disease (3, 10, 22–26). Other imaging techniques, such as optical coherence tomography (OCT) and confocal laser endomicroscopy (CLE), have also shown promise for the detection of early-stage laryngeal carcinoma (27–30). The benefit of the method lies in the evaluation of the microarchitecture of the epithelium and lamina propria. OCT enhances the optical imaging of the entire thickness of epithelium and the basement membrane, which is critical in the assessment of invasiveness (28, 31). CLE imaging using fluorescein as a contrast agent allowed the visualization of the tissue architecture at a cellular level with up to x1000 magnification, resulting in additional dimension for healthy mucosa and squamous cell carcinoma differentiation contrast to NBI/IMAGE1 S endoscopy (28, 30). They can be especially helpful in the evaluation of non-transparent flat lesions, as leukoplakias or erytroplakias (28, 29), but also in assessment of the IPCLs of the normal mucosa and squamous cell carcinoma (32).

As a first Piazza et al. presented HDTV with NBI endoscopy focused on laryngeal lesions as method with high sensitivity (98%) and specificity (90%) in diagnosis malignant lesions compared to white-light endoscopy only (24). According the contemporary literature any well-demarcated brownish area with thick dark spots and/or winding vessels was judged as a “suspicious” lesion at NBI (22). Piazza et al. analyzed these “suspicious” lesions and carcinoma in situ or invasive carcinoma were histologically confirmed in 86.2% (24)..

The lack of adequate classification of neoplastic and non-neoplastic lesions was one of the most important flaws of existing NBI literature (33). Different levels of dysplasia being counted as “positive,” there is a lack of consistency as to what constitutes NBI positive results. Even though many studies reported a “positive” endoscopy if a well demarcated brownish area with scattered brown spots is seen, the interpretation of this in the context of an endoscopy is going to be subjective for the examiner and based on experience (34–36). There was little attempt made to account for interoperator variability.

A clear description of vascular architecture and the first classification based on the IPCL patterns in the larynx was published by Ni et al. (5). The proposed descriptive classification is probably the most well-documented classification system of these vascular changes. It has been used in numerous studies of laryngeal and pharyngeal lesions and has improved the diagnostic accuracy of NBI (3, 9, 15, 33, 37–41).

Regular oblique vessels and dendritic vessels (vascularization type I) or arborescent vessels with enlarged diameter (vascularization type II) correspond to benign lesions in 100% and 87.5–100% of cases, respectively (5, 9). On the other hand, the damaged, expanded, and distorted IPCLs with their irregularity and extension to the twisted line shape, often progressing to abnormal vessels with uneven density (vascularization type V), are characteristic of invasive carcinoma in 86.5–91.6% of cases (9, 42). Consequently, correct estimation of the histopathological diagnosis of these types of vascular features during the endoscopic examination is not problematic.

Borderline cases present visible, relatively regularly arranged, scattered, small, dark brown spots of low density, the dot-like IPCLs. IPCLs of type IV, according to Ni et al., occupy a “gray” area of interpretation of neoangiogenesis. In their original study they described this vascular pattern in 19/104 patients who were histologically mostly diagnosed with hyperplasia and mild dysplasia. The authors considered these to be IPCLs rather than a benign type of vascular loops, but the number of enrolled patients in their study was low (5). On the other hand, the European Laryngological Society (ELS) classification published by Arens et al. describes dot-like changes as perpendicular vascularization with malignant potential (7). Non-uniformity of classification, especially their views on “brown spots” or the symmetric intraepithelial capillary loops, lead to less accurate prediction of malignant transformation of these lesions in clinical praxis (11, 43).

Popek et al. analyzed 28 suspicious lesions with the type IV vascularization in the group of 333 patients with laryngeal lesions. In 17/28 cases they were classified as mild or moderate dysplasia, and 1/28 case was confirmed severe dysplasia or invasive carcinoma (40). Berlito et al. published a study of 248 patients, and the type IV lesions (12/248, 4.8%) corresponded in 100% of cases to benign and/or preneoplastic lesions (mild or moderate dysplasia) (9). Rzepakowska et al. reported a group of 91 laryngeal leukoplakia cases, which included 11/91 lesions with type IV vascularization with histological confirmation of parakeratosis or low-grade dysplasia (44). Lu et al. declared in their study of 119 patients that epithelial proliferation and low-grade (mild and moderate) intraepithelial neoplasia were mostly classified as having type III and IV vascularization (84.2%), which were significantly more common than other Ni classification subtypes. However, in the same study, 7/25 lesions with type IV vascular networks were histologically verified high-grade intraepithelial neoplasia, carcinoma in situ, or invasive carcinoma (42). Ahmadzada et al. published the results of an NBI evaluation of laryngeal cancer. They found that 10/12 cases of type IV vascularization were severe dysplasia, and 2/12 false negative cases were carcinoma in situ and invasive carcinoma (45). A limitation of existing studies is the number of analyzed lesions with type IV vascular patterns. Our study presents 293/674 (43.5%) lesions with type IV IPCLs, and therefore represents several times more examinations than have been reported in previously published studies. According to our results ability to recognize benign lesions in group of type I-IV vascular patterns, which are connected with non-malignant lesions, and distinguish them from precancers and cancer lesions is with low sensitivity (54.4%) and low negative predictive value (69.9%). Analysis of histopathological results in group of uncertain vascular changes (type IV vascularization) detected large portion of dysplastic changes (106/293, 36.2%) and these changes represented 106/158 (67.1%) of all dysplastic lesions in examined cohort. Low grade dysplasia was detected in 20.8% and high grade dysplasia in 15.4% lesions with type IV vessels. Type IV vascularization should be considered a marker for precancerous lesion, and thus it cannot be considered a benign or non-malignant type of vascularization.

Depending on the study, the different levels of dysplasia have been classified as “positive” or “negative,” and there is a lack of consistency as to what constitutes NBI-positive results (33, 46). It is presumed that laryngeal dysplasia has a higher rate of malignant transformation than normal epithelium (47). Van Hulst et al. specifically studied the correlation between the grade of dysplasia and development of invasive laryngeal cancer in a systematic review of the medical literature, and they concluded that rates of malignant transformation in mild dysplasia range from 0 to 41.7%, in moderate dysplasia from 0 to 48.0%, in severe dysplasia from 14.3 to 44.4%, and in carcinoma in situ from 11.1 to 75% (48)..

In contrast, a study of 70 patients during a 10-year period published by Luers et al. concluded that the risk of developing laryngeal squamous cell carcinoma out of laryngeal dysplasia shows no statistical correlation to the initial dysplasia grade (49). The patients with laryngeal dysplasia are an inhomogeneous group and, as stated above, the grade of laryngeal dysplasia alone seems to be an insufficient prognostic factor for the development of laryngeal cancer.

According to a recently published review of current knowledge, ELS considers the diagnosis of laryngeal dysplasia to largely rely on endoscopic procedures and histopathology. The diagnostic efficiency of endoscopy may be improved using videolaryngostroboscopy (VLS) and bioendoscopic tools such as NBI or IMAGE1 S (50). Current histological classifications are not powerful enough to clearly predict the risk to carcinoma evolution, and technical issues such as sampling error, variation in epithelial thickness, and inflammation hamper pathological examination (50). However, different studies have defined the possible utility of different biomarkers: cell proliferation factor Ki-67, cell cycle control (p 53, p16), transcription factor of embryonic stem cell pluripotency (NANOG), chromosome instability, and mutational profiles have been able to predict this risk more accurately and have demonstrated their practical utility (51–56). These methods are still in development and are not commonly used in clinical practice. Combining the assessment of IPCL patterns with molecular marker analysis might further improve the diagnostic accuracy and prognostic value of enhanced endoscopic methods.

In our study, benign lesions were detected in 178/293 (60.7%) and low-grade (mild) dysplasia in 61/293 (20.8%) of cases with type IV vascularization. In 78.2% of all mild dysplasia are these lesions represented by type IV vascularization. Especially, the high negative predictive value certifies NBI to be an excellent tool for the follow-up of certain cases of premalignant lesions with mild dysplasia, thus avoiding unnecessary biopsies or surgeries (46).

High-grade (moderate, severe and carcinoma in situ) dysplasia or SCC were confirmed in 54/293 (18.4%) of lesions categorized as having type IV vascularization in our group of patients. The risk of malignant transformation of severe dysplasia and carcinoma in situ to SCC is higher than in low-grade dysplastic lesions (48). This is the reason to examine the other features of the vascular network or the macroscopic appearance of the lesions to better clarify the character of vascular changes in laryngeal dysplasia.

Šifrer et al., in the study of 80 vocal cords in 40 patients, described the impact of distinguishing between narrow- and wide-angled turning points of the IPCL in differentiating between carcinoma and papilloma, respectively (43). In the other study Šifrer et al. confirmed the effectivity of ELS classification in correct interpretation of perpendicular vascular patterns with accuracy 95% (12). Lukeš et al. concluded that the most important observation for a correct diagnosis is change in IPCL shape. In distinguishing between SCC and papillomatosis, it is crucial to focus on the epithelial surface and to observe the papillary structures with a central-axis capillary and a more or less regular arrangement. Some of the associated endoscopic characteristics, such as the surface, the presence of multiple lesions, and the spread to both vocal cords, may be used as criteria for a more accurate diagnosis (25). Rzepakowska et al. presented NBI accuracy with high sensitivity and specificity (100% and 97.4%) in stratifying the risk of malignant lesions in vocal cord leukoplakia by evaluating the vessels in the epithelium surrounding the white plaque (44).

NBI endoscopy seems to be a perfect tool for the evaluation of vascular changes in the laryngeal epithelium (50, 57). However, adequate interpretation of “optical” biopsy lesions with type IV vascularization according to Ni is difficult because of the considerable variability of final histopathology results. Studies published in recent years confirm that using NBI combined with contact endoscopy to detect high-grade dysplasia and carcinoma increases diagnostic quality (13, 58).

In the future development of computer-aided diagnostic systems and investigation of computer analysis of endoscopic NBI images may further improve the quality of diagnosis (59, 60). Recent studies have demonstrated the potential of machine learning algorithms for the classification of vascular patterns in differentiating between benign and malignant lesions (59–62). Interobserver variability in the interpretation of NBI findings was one of the reason to introduce vascular classification. Study of Mehlum et al. showed that the 5-tier classification of Ni et al. (5) and Puxxed et al. (6) makes it difficult to reliably classify vascular changes in contact endoscopy and NBI even for experienced ENT surgeons. On the other hand, use of the two-level classification recommended by the ELS significantly improves the differentiation of non-cancerous lesions from cancerous lesions (63). Difficulty of clear classification the lesions is a potential limitation in the clinical application of this technique. This finding may improve the validation of IPCLs, as well as the use of artificial intelligence, might help minimize interobserver variability and improve diagnostic accuracy (60, 62).

Laryngeal lesions with type IV vascularization, as defined by Ni, present various histological findings, and include several precancerous and malignant lesions. Patients with type IV vascularization must be followed carefully. When mucosal lesions progress, microlaryngoscopy and excision are indicated.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving humans were approved by Ethic Committee of University Hospital Ostrava. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin.

LS and KZ contributed to the study’s conception and design and wrote the manuscript. LS, PKá, and KF performed the data collection and analysis. KZ and PKo supervised this study. All authors contributed to the article and approved the submitted version.