Pathology of Nonneoplastic Lesions of the Vocal Folds

Nonneoplastic vocal fold lesions are common that can cause hoarseness and voice change. Reactive lesions of Reinke’s space can be observed in all ages and genders and comprise the majority of the benign nonneoplastic vocal fold lesions. Although clinically different terms are used to define reactive lesions of Reinke’s space, they share the same histopathologic features. In order to differentiate vocal fold polyp and nodule and Reinke’s edema, clinical findings should be considered. Epithelial changes such as pseudoepitheliomatous and verrucous hyperplasia may cause diagnostic challenge due to resemblance of squamous cell carcinoma. Evaluation of the invasion border and cellular atypia may aid in correct diagnosis.


Introduction
The vocal folds, also known as vocal cords, extend through the laryngeal cavity bilaterally and are primarily responsible for voice production. The thyroarytenoid muscle, deep lamina propria, intermediate lamina propria, superficial lamina propria, and squamous epithelium comprise the vocal folds [1-3]. The superficial lamina propria, which is also called Reinke's space, consists of loose fibrous or elastic components and provides a gelatinous surface for vocal folds to vibrate upon. The deep and superficial lamina propria forms the vocal ligament. Madruga et al. demonstrated that type I collagen is abundant in the superficial lamina propria which forms a narrow band. The intermediate lamina propria essentially contains type III collagen, and the deep lamina propria is rich from both type I and type III collagen [4].

Vocal fold nodule and polyp
Vocal fold nodules and polyps are polypoid lesions of the vocal folds sharing the same histopathologic features. They are usually differentiated by clinical findings. A polypoid lesion larger than 3 mm is often regarded as a polyp, whereas smaller lesions are classified as a nodule [31]. Vocal fold nodules are usually bilateral lesions involving anterior or middle vocal fold and located superficially to the free edge of the vocal fold [32]. They are slightly more common in women and in young age [10,33]. Vocal polyps occur more commonly at the anterior portion of the vocal fold, and in more than 90% of the patients, they are unilateral [31]. There is no age and gender predilection for vocal polyps. Vocal abuse is the leading etiologic factor for vocal fold nodules and polyps; singers, lecturers, and coaches are more prone to developing these lesions due to excessive and loud voice use [6,10]. Infection, hypothyroidism, GERD, cigarette smoking, and allergy are the other frequent causes [7][8][9]34]. Patients present with hoarseness and change in voice quality.
Grossly, vocal cord polyps and nodules present as sessile or polypoid lesions with nodules presenting as few millimeters and polyps up to few centimeters in size [5]. They can be white or bright red, and their consistency varies from firm to soft with a mucoid or glistening cut surface [32]. Although previous studies have attempted to identify histopathologic features which may aid in the differential diagnosis, it is now widely accepted that vocal fold nodules/polyps cannot be differentiated on the histopathologic basis [11,31,32,35]. Epithelial hyperplasia, basement membrane thickening, edema, and vascular proliferation can be seen both in nodules/polyps and Reinke's edema [25,31,36]. Ancillary studies such as Verhoeff-van Gieson, Masson trichrome, and Alcian blue did not reveal any difference among these entities [31].
Histopathology represents primarily the extent of the vascular damage and the temporal stage of the lesion [37]. Vocal abuse causes vascular injury and increased vascular permeability. If the damage is minimal, then increased permeability causes only edema, and the microscopic evaluation reveals hypocellular myxoid stroma which is defined as edematous myxoid-type vocal fold nodule/polyp according to some authors (Figure 1). When this lesion is not removed or resolved, it undergoes fibrosis, and fibrous-type polyp/nodule evolves. Oval and spindle cells are observed embedded in a fibrous stroma (Figure 2). Rarely atypical cells can be detected in this type which is not related with an aggressive behavior [36,38,39]. However, if the vascular injury is severe enough for fibrin escape to the subepithelium and interstitium at the beginning of the injury, then hyaline-type polyp/nodule occurs which is characterized by a hypocellular/acellular eosinophilic stroma. By the time capillary proliferation occurs, it progresses to a vascular-type nodule/polyp. This subtype shows ectatic vascular channels in a hypocellular stroma (Figure 3). Although, these classification and staging are helpful in understanding the  histopathologic spectrum, it is clinically insignificant. In addition, mixed features are seen in a single lesion most of the time. The overlying squamous epithelium may be normal, atrophic, or hypertrophic, and keratosis can be seen.
Differential diagnosis includes amyloidosis, myxoma, and contact ulcer. Differentiation of hyaline-type vocal fold nodule/polyp from amyloidosis can be made by observing the distribution of eosinophilic material carefully. In hyaline-type nodule/polyp, it is more homogenous, whereas in amyloidosis there is perivascular and periglandular accentuation of the deposit. Histochemical stains like Congo red and crystal violet can be helpful in identifying the nature of the amorphous material. Myxoma has been rarely reported in the larynx, and it has characteristic stellate spindle cells embedded in an avascular, basophilic, gelatinouslike matrix [40,41]. Contact ulcer has an ulcerated and fibrin-coated surface and usually involves the posterior portion of the vocal folds.
Surgery has a limited value for these lesions as they are reactive changes to an underlying injurious condition which must be managed. Not surprisingly, voice therapy may result in resolution in most of the patients [42,43]. Also, treatment of the exact underlying condition such as hypothyroidism can be helpful in the treatment of vocal fold polyps/nodules.

Contact ulcer
Contact ulcer is an ulcerated granulation tissue due to vocal abuse, endotracheal intubation, and GERD. Men are more commonly affected than women except that postintubation-related ulcers are more common in female patients. Clinically patients present with hoarseness, cough, dysphonia, habitual coughing, and throat cleaning [44].
Contact ulcers are generally bilateral ("kissing ulcer") and involve the posterior part of the vocal fold. Grossly they present as an ulcerated, tan-white to erythematous polypoid lesion up to 3 cm in diameter. Microscopic evaluation reveals ulceration underlying a fibrin and/or fibrinoid necrosis and capillary proliferation that is oriented perpendicularly to the mucosal surface (Figure 4). Central and base part of the lesion contains hemosiderin-laden macrophages. Usually a dense inflammatory infiltration accompanies. Re-epithelization, mucosal hyperplasia, and fibroblastic proliferation can be observed in chronic phase of the lesion [45].
Clinicopathologic correlation is important in the correct diagnosis as histopathologic features are somewhat nonspecific. Differential diagnosis includes vasculartype vocal cord polyp/nodule, Kaposi sarcoma, Wegener granulomatosis, and infectious agents. Wegener granulomatosis can be differentiated from contact ulcer with features of vasculitis and necrotizing granulomas. Also, laboratory findings, such as cytoplasmic antineutrophil cytoplasmic antibody (c-ANCA) positivity, support a diagnosis of Wegener granulomatosis over contact ulcer. Kaposi sarcoma may rarely involve the larynx, and histopathologic evaluation reveals spindle cells with intracellular spheroid hyaline globules forming slit-like spaces and anastomosing vascular channels. Immunohistochemical staining with human herpes virus 8 (HHV8) reliably confirms the diagnosis as Kaposi sarcoma.
Like vocal polyps/nodules, contact ulcer is treated with voice therapy, anti-GERD medication, and behavioral modifications in order to decrease habitual coughing and throat clearing [44].

Amyloidosis
Amyloidosis is the deposition of an extracellular, acellular, fibrillar, and amorphous material in various sites of the body. It can be primary (associated with plasma cell neoplasms) or secondary (associated with chronic systemic diseases such as familial Mediterranean fever, rheumatoid arthritis, etc.). Primary amyloidosis can be classified as systemic or localized. The larynx is the most common site for localized amyloidosis [46,47]. It may also be associated with "mucosa-associated lymphoid tissue" (MALT) lymphoma or a neuroendocrine tumor. Laryngeal amyloidosis generally affects adults at the sixth decade. Patients present with hoarseness and voice changes because of mass effect [45]. Grossly it presents as a firm polypoid lesion up to 4 cm covered by normal-appearing mucosa. Cut surface may be firm and starch-like and gray or yellow-orange in color. Localized laryngeal amyloidosis can involve the larynx diffusely or present as a nodule. Microscopic examination reveals diffuse subepithelial or discrete nodular lesion consisting of amorphous, fibrillary, and hyaline-like eosinophilic substance deposition. Usually the distribution of the deposition is more pronounced in perivascular and periglandular areas. Lymphoplasmacytic infiltration and foreign body giant cells can be seen but usually sparse.
Differential diagnosis includes hyalinized-type vocal fold polyps/nodules and lipoid proteinosis which are negative with Congo red and crystal violet. Most importantly as amyloid can be associated with multiple myeloma, laryngeal neuroendocrine tumors, and medullary thyroid carcinoma, a systemic evaluation of the patient is necessary. If amyloidosis is limited to the larynx, patients are treated with surgical removal of the lesion. However, other treatment options will be applied if the patient has an underlying neoplastic condition or chronic inflammatory disease.

Laryngocele
Laryngeal ventricles are the spaces between the true and false vocal cords which extend upward and form the laryngeal saccule bilaterally. Laryngocele can be defined as a symptomatic dilation of the laryngeal saccule with air entrapment [51]. An important feature of laryngocele is that the lumen of the cyst communicates with the laryngeal cavity. Radiography reveals an air-filled cystic enlargement. They are usually unilateral, may occur over a wide age range, and are more frequent in males. Occupations such as glassblower or wind instrumentalists are at increased risk of developing laryngocele due to repetitive increase in intralaryngeal pressure. Patients present with hoarseness and foreign body or globus sensation.
Clinically it can be divided as internal, external, or combined. Internal laryngocele is confined to the endolarynx and presents as a supraglottic submucosal lesion. In contrast external laryngocele extends through the thyrohyoid membrane. However, in combined laryngocele, the cyst herniates through the thyrohyoid membrane and presents as an anterior neck mass [51][52][53]. Patients present with hoarseness, dyspnea, and chronic cough. In histopathologic examination, the respiratory epithelium is identified as the lining of the cyst wall.
Differential diagnosis includes other laryngeal cysts. However, laryngocele is the only air-filled cyst of this region and communicates with the laryngeal cavity-an important finding in differentiation.
Symptoms may resolve with the expulsion of trapped air from the cyst lumen. Treatment of laryngocele is simple excision or marsupialization of the cyst wall.

Saccular cyst
Saccular cyst is a mucin-filled cyst due to obstruction of the laryngeal saccule. It can be acquired or congenital [49,50]. If it extends medially, it may obscure the anterior vocal fold. Lateral saccular cysts are similar to the external laryngoceles, herniated through the thyrohyoid membrane, and may present as a neck mass [37]. Saccular cysts are usually lined by respiratory-type epithelium. Squamous or oncocytic lining can be observed in some cases. Cyst lumen is filled with mucin or acute inflammatory exudate.
Differential diagnosis includes laryngocele and thyroglossal ductus cyst. It is differentiated from laryngocele by mucin content, and it does not communicate with the laryngeal cavity. It is difficult to discriminate a thyroglossal cyst from a saccular cyst when it invades through the pre-epiglottic space and histopathologically lacks the thyroid tissue. When this is the situation, investigation of the anatomic localization of the cyst is a reliable finding in the differentiation of these cysts. Thyroglossal ductus cysts are related with the hyoid bone and located in the midline of the neck.

Ductal cysts (squamous, tonsillar, oncocytic)
Ductal cysts are the most common type of laryngeal cysts. Cyst lining epithelium can be squamous or oncocytic [49,54] (Figures 5 and 6). When a squamous lined cyst is surrounded by lymphoid tissue, it is called a tonsillar cyst. Tonsilar cysts are more common in the vallecula which contains tonsillar remnants.

Other cysts
Rarely epidermal, dermoid, and branchial cleft cysts may occur in the endolarynx.

Hyperplastic lesions of the larynx
Hyperplastic lesions of the larynx are a result of a reparative process. Clinical terms such as leukoplakia (white plaque), erythroplakia (red plaque), erythroleukoplakia (red and white changes), or pachydermia (extensive thickening of the mucosa) are used to describe the lesion macroscopically, and they are not histologic entities [55].
Keratosis of the larynx is synonymously used with simple hyperplasia and squamous hyperplasia [56]. Singers and other occupations that lead to an individual to use his/her voice excessively and cigarette smokers are at risk of keratosis. Hoarseness is the leading complaint. Laryngoscopy identifies leukoplakia. Microscopic evaluation reveals a thickened squamous epithelium without cellular or architectural atypia [55]. Generally, a hyperkeratotic layer overlies the epithelium. Lesions with warty configuration are referred as verrucous keratosis [56,57].
Pseudoepitheliomatous hyperplasia (PEH) is a reactive proliferation of the squamous epithelium which develops in response to infections, trauma, or neoplasia. Histologically these lesions are downward thickening of the epithelium (Figure 7). Due to a less well-defined epithelial stromal interface and the tendency of anastomosing epithelial tongues entrapping the submucosa, the lesion may mimic squamous cell carcinoma [58]. However, in PEH, the hyperplastic epithelium forms large bulbous projections, and basement membrane is always preserved.  Verrucous hyperplasia is a verrucous and keratotic form of squamous hyperplasia which may show varying degrees of cytological atypia (Figure 8). The presence of cellular atypia and a relatively regular epithelial-stromal border supports a diagnosis of verrucous hyperplasia over verrucous squamous cell cancer (SCC) [59].

Conclusions
Voice change or hoarseness of voice and nonneoplastic vocal fold lesions lead to a high percentage of vocal fold biopsies. Both clinical and histopathologic features should be considered in order to differentiate lesions involving Reinke's space and also hyperplastic epithelial lesions from squamous cell carcinoma.