Update from the 5th Edition of the World Health Organization Classification of Head and Neck Tumors: The Neck and Lymph Nodes, Metastasis, and Melanocytic Tumors

Abstract

The changes made in the fifth edition of the WHO Classification of Head and Neck Tumors demonstrate the recent diagnostic, histopathological, and molecular advances in the field, and this updated information will hopefully lead to improved and standardized tumor subtyping. This review summarizes the changes related tumors and tumor-like lesions of the neck and lymph nodes (Chapter 11), metastasis to the head and neck region (Chapter 15), and melanocytic tumors (Chapter 10).

Introduction

The fifth edition of the WHO Classification of tumors of the Head and Neck Tumors—also called the “WHO Blue Book”—has incorporated changes in its discussion of tumors and tumor-like lesions of the neck and lymph nodes (Chapter 11). In addition, a new chapter devoted to the discussion of metastasis to the head and neck region (Chapter 15) and a new chapter focused on mucosal-based melanocytic tumors (Chapter 10) are now part of the table of contents. This article will summarize the changes related to new classification regarding these three chapters since the 2017 edition, highlighting essential and desirable diagnostic histopathological and molecular criteria, along with new entries (Table 1).

Table 1.

Essential and desirable diagnostic criteria of tumors and tumor-like lesions of the neck and lymph nodes

	Essential	Desirable
Cysts and cyst-like lesions
Ranula	Cyst-like structure containing mucin surrounded by a fibrous or granulation tissue, without an epithelial lining	Imaging: Extension of the lesion into the submandibular space (tail sign)
Lymphoepithelial cysta	Dilated cystic structure, benign epithelial lining, dense lymphoid stroma in cyst wall
Branchial cleft cyst	Lateral neck location, bland cytomorphology, negative HPV status
Thyroglossal duct cyst	Perihyoidal location	Midline respiratory or squamous lined cyst with associated thyroid follicular epithelium
Dermoid cyst	Squamous epithelial-lined cystic lesion with associated adnexal structures, to possibly include hair follicles and glandular elements (sebaceous, eccrine, and/or apocrine)
Teratoid cyst	Same as above but including endodermal tissues such as respiratory or gastrointestinal mucosa
Other tumors
Branchiomaa	Solitary, well circumscribed mass located in suprasternoclavicular area Haphazard proliferation of benign spindled cells, epithelial islands, and adipocytes, in the absence of primitive elements
Heterotopia-associated carcinoma	Carcinoma arising in a known location for heterotopic tissue, clinical and/or pathologic exclusion of a prior or concurrent orthotopic carcinoma	Adjacent benign heterotopic tissue
Carcinoma of unknown primary (CUP)	(True CUP) p16 and EBER negativity, and no primary site identified after extensive diagnostic work-up and clinical history	(If test is available) Lack of UV-signature mutations

^aNew entry in the 5^th Edition of World Health Organization (WHO) Classification of Tumors of the Head and Neck

Tumors and Tumor-Like Lesions of the Neck and Lymph Nodes

The neck is a complex anatomic region that encompasses lymph nodes, vascular and lymphatic structures, nerves, and various types of soft tissues. As proposed by the American Head and Neck Society and the American Academy of Otolaryngology–Head and Neck Surgery, the topographic anatomy of the neck can be divided into 6 levels (Fig. 1) [1]. In the daily practice of head and neck pathology, it is imperative to know the lymph node level designations of the neck, as anatomic location can provide diagnostic clues, particularly in the setting of metastatic disease and in the evaluation of cystic lesions. Therefore, the fifth edition reiterates the importance that clinicians provide the exact location of the lesion in question as opposed to labeling a specimen as a neck mass.

Fig. 1.

The location of cervical lymph nodes in the neck follows a system that divides the topographic anatomy of the neck in six levels: IA (submental nodes), IB (submandibular nodes), IIA and IIB (together comprising the upper jugular nodes), and VA (spinal accessory nodes) and VB (transverse cervical and supraclavicular nodes). (Image by Jill K Gregory, CMI, Icahn School of Medicine at Mount Sinai)

Since the fourth edition, the WHO has recognized the need to include a separate chapter in the Blue Book to discuss tumors and tumor-like lesions affecting the neck. In the fifth edition, this section has been reorganized into two groups: (1) cysts and cyst-like lesions and (2) other tumors. Notably, lymphoepithelial cysts and branchioma are now included in this chapter. In contrast to the fourth edition, hematolymphoid tumors and Merkel cell carcinoma are excluded from this chapter and instead discussed at length in other sections of the book. Other cystic lesions of the neck with distinct clinical and histological features are still absent, including lymphangioma, cervical thymic cyst, external laryngocele, and cervical bronchogenic cyst.

Cysts and Cyst-Like Lesions

Cystic lesions of the neck are commonly encountered in clinical practice, and understanding their presentation, anatomical location, and histopathological evaluation of the cyst lining are key in rendering an accurate diagnosis.

Ranula

A ranula is a pseudocyst secondary to the extravasation of mucin, and its name is derived from the Latin word rana because of its macroscopic similarity to the belly of a frog [2]. Oral ranulas, also referred as simple ranulas, present as painless, translucent-to-blue, dome-shaped cystic lesions affecting the floor of the mouth and are associated with the sublingual gland. In some cases, the lesions protrude along the posterior border of the mylohyoid muscle or through a defect in the muscle and present as a cervical submandibular space mass, a phenomena referred as a plunging ranula [2]. On diagnostic imaging, plunging ranulas may demonstrate the so-called tail sign, which consists of a smooth tapering comet-shaped unilocular fluid mass with its “tail” in the collapsed sublingual space and its “head” in the submandibular space [3]. The histopathologic features of oral and plunging ranulas are similar and include a cyst-like structure surrounded by fibrous tissue, that is typically devoid of an epithelial lining, contains mucin, and is associated with granulation tissue and histiocytes (mucophages) [4].

Branchial Cleft Cyst

Overall, branchial cleft lesions are considered developmental anomalies secondary to incomplete involution of branchial cleft structures during embryogenesis and can be classified as sinuses, fistula, and cysts. Over 95% of branchial cleft cysts represent anomalies of the second branchial cleft apparatus [5]. Patients typically present with a unilateral painless mass in the lateral neck along the anterior surface of the sternocleidomastoid muscle [5]. Among patients with upper respiratory tract infections, the size of the cyst can suddenly increase.

Morphologically, branchial cleft cysts are usually unilocular and show a bland epithelial lining, mostly stratified squamous epithelium, and associated with a dense lymphoid stroma in the cyst wall (Fig. 2). Acute inflammation associated with fibrosis and granulation tissue may be seen if the cyst has ruptured. In patients aged ≥ 40 years particularly presenting with a level II/III mass, it is essential to exclude a cystic lymph node metastasis from an HPV-associated oropharyngeal squamous cell carcinoma [6]. This distinction can be made based on the absence of marked atypia epithelial lining in branchial cleft cyst and careful use ancillary testing.

Fig. 2.

Branchial cleft cyst. A Solitary right level II unilocular cystic lesion on CT scan; B On scanning magnification, a well-delineated cystic is apprenticed with a dense lymphoid stroma in the cyst wall; C The cyst is lined by bland stratified squamous epithelium; D The cyst lining can show p16 reactivity; E RNA in-situ hybridization for high-risk HPV is negative

The fourth edition of the Blue Book alerted readers to the limitations of p16 staining in branchial cleft cysts, as approximately 30% to 40% of cases can show reactivity for this marker [7, 8]. To avoid this pitfall, the fifth edition suggests using high-risk HPV-specific testing, such as polymerase chain reaction (PCR) based assays or RNA in-situ hybridization [9]. However, it is important to note that the p16 expression in branchial cleft cysts is usually limited to the superficial epithelial lining cells and typically the extent of staining does not exceed 40%, which is below the 70% threshold required by College of American Pathologists (CAP) to define a sample as HPV-positive [10, 11]. In addition, the fifth edition reiterates that the overwhelming majority of primary branchiogenic carcinoma reported in the past most likely represent metastatic cystic HPV- associated squamous cell carcinoma [12].

Lymphoepithelial Cyst

In the fourth edition of the 2017 Blue Book, the term lymphoepithelial cyst was listed as a synonym of branchial cleft cyst, but in the current classification, lymphoepithelial cyst is discussed as an independent entity. This distinction is debatable because of the overlapping morphology of the 2 lesions; however, the fifth edition states that lymphoepithelial cyst is a descriptive term that encompasses a variety of acquired cystic lesions that share histological features but occur in different clinical settings (i.e., immunocompetent individuals, Sjögren syndrome and HIV-related sialadenitis) and anatomic locations (i.e., parotid, angle of mandible, lateral neck, and oral cavity). As its name implies, a lymphoepithelial cyst consists of a dilated cystic structure with benign epithelial lining that is associated with a dense lymphoid stroma and frequently permeated by mature lymphocytes (Fig. 3). The utility of recognizing lymphoepithelial cyst as a standalone diagnostic category remains open to discussion.

Fig. 3.

Lymphoepithelial cyst. A Dilated cystic lesion with prominent lymphoid stroma; B Epithelial lining shows a squamoid appearance and lacks cytomorphological evidence of malignancy; C In the setting of HIV-related sialadenitis, lymphoepithelial cyst characteristically shows lymphoepithelial lesions in the adjacent lymphoid stroma

In the setting of HIV infection, the cysts affect intra- or periglandular lymph nodes. The background lymphoid tissue exhibits the histologic spectrum of features observed among patients with AIDS-related persistent generalized lymphadenopathy, ranging from bizarre florid follicular hyperplasia to follicle lysis [13–15]. Scattered multinucleated giant cells localized to the interfollicular areas or close to the epithelial cysts are frequently seen. Those giant cells, as well as follicular dendritic cells, demonstrate immunoreactivity for the HIV-1 major-core protein (p24) [16]. The cysts show a squamous epithelium, with different degrees of maturation and permeation by mature lymphocytes. In addition, another feature of HIV-related sialadenitis includes the presence of irregular solid squamoid epithelial nests embedded in an intense lymphoid stroma, called a lymphoepithelial lesion [17]. The latter is also a histologically found in lymphoepithelial sialadenitis (LESA), which is commonly associated with Sjögren syndrome and other autoimmune conditions. LESA can also exhibit cystic changes, but these are typically not as pronounced as those in cases of HIV-related sialadenitis [18].

Thyroglossal Duct Cysts

Thyroglossal duct cysts are derived from a failure of obliteration with subsequent dilation of the thyroglossal duct, which is an embryological structure that directs the migration of the thyroid gland primordium from the foramen cecum to its final position in the anterior midline of the neck [19]. Therefore, thyroglossal duct cysts are midline lesions, and the majority of patients with these lesions present with a painless anterior neck mass at the level of the hyoid bone that moves with deglutition. Unusual locations include the base of the tongue and lateral neck among patients with prior surgery or recurrent infections [20]. The Sistrunk procedure, which consists of the excision of the cyst, its associated suprahyoid fibrous tract, and the central portion of the hyoid bone, is the treatment of choice because it yields low rates of recurrences [21].

The lining of thyroglossal duct cysts varies from columnar respiratory to predominantly nonkeratinizing stratified squamous epithelium, and approximately half of cases show a combination of both. Morphological features of cyst rupture, including foamy histiocytes, cholesterol clefts, and a foreign body giant cell reaction, are common. In a subset of cases, no epithelial lining is identified, and the lesion only consists of a fibrous cyst wall. The amount of ectopic thyroid follicular tissue in the cyst wall and/or the fibrous tract varies, and in 30% to 40% of lesions, it can be absent [20]. In such scenarios, the diagnosis of thyroglossal duct cyst is established based on clinical presentation, anatomic location, and intraoperative findings. Malignant transformation of the ectopic thyroid follicular tissue may occur, and this phenomenon will be discussed in “Heterotopia-Associated Carcinoma” section.

Dermoid Cysts

Dermoid cysts are developmental cystic lesions that are derived from entrapped ectodermal and mesoderm elements along planes of embryonic fusion; these cysts present congenitally or during the first 5 years of life [22]. They affect the subcutaneous tissue and can be found anywhere on the body, with the vast majority occurring in the head and neck region. At this location, the distribution commonly includes the outer third of eyebrow, the scalp, the dorsum of the nose, the eyelid and the anterior neck [23]. Dermoid cysts affecting the midline at the scalp and the dorsum of the nose might be associated with cranial dysraphism and intracranial extension [24]. Histologically, the lesions are lined by stratified squamous epithelium with skin adnexal elements present in the connective tissue cyst wall. Keratinaceous debris and hair can be identified the lumen. Lesions called teratoid cysts also contain endodermal derivatives in the cyst wall, such as respiratory or gastrointestinal mucosa, as well as mesoderm derivatives including muscle and cartilage [25].

Branchioma

One of the most notable updates in this edition of the WHO classification to the chapters about tumors and tumor-like lesions of the neck is the debut of branchioma. This tumor was first described in 1982 by Smith and McClure, who provisionally regarded it as an unusual mixed tumor featuring mesenchymal and epithelial components affecting the supraclavicular fossa [26]. Two years later, Rosai et al. coined the term ectopic hamartomatous thymoma to describe five similar cases located in the soft tissues of the lower neck among adults and characterized by the presence of 4 components: spindle cells, epithelial islands, mature adipose tissue, and lymphocytes (Fig. 4) [27]. It was believed that these unusual findings indicated a benign tumor that was derived from the third branchial arch and was composed of abnormal thymic tissue. Since then, the histogenesis this tumor has been a source of debate, as well as its nomenclature. Consequently, other terminologies have been proposed, including branchial anlage mixed tumor, thymic anlage tumor, and biphenotypic branchioma [28–30]. The term biphenotypic branchioma is endorsed by the WHO Blue Book based on the consensus that this lesion is a true neoplasm derived from the branchial apparatus and is not related to thymomas or salivary gland mixed tumors (pleomorphic adenomas). Because the branchial apparatus contributes to the formation of the neck, the tumor should not be considered ectopic, and the acceptable designation biphenotypic refers to presence of different tissue types within lesion, presumptively of mesodermal and endodermal origin.

Fig. 4.

Biphenotypic branchioma. A The histological hallmark of biphenotypic branchioma is the presence of spindle cells, epithelial islands, and mature adipose tissue; B the epithelial islands most commonly show non-keratinizing morphology (Images courtesy of Dr Annikka Weissferdt, Department of Pathology, MD Anderson Cancer Center, Houston, TX)

Branchiomas are rare tumors, with less than 100 cases reported in the English literature. Middle-aged adults (age range, 19–89 years; predilection mean, 46 years) are most commonly affected with this tumor, with significance male predominance [30]. The tumors typically occur in the deep lower anterior neck region, most commonly in the supraclavicular area, followed by the presternal and sternoclavicular region. Interestingly, the left side of the neck is more commonly affected. Rare cases involving the axilla and chest wall have been reported [31, 32]. Branchiomas usually presents as slow-growing, painless masses, and conservative but complete surgical resection is curative. Recurrent disease is rare, and no metastases or tumor-related deaths have been reported [27–30].

Branchiomas are well-demarcated, with variable encapsulation, and showing a solid and white to yellowish cut surface; reported tumor size ranges from 2 to 19 cm [29, 30]. The morphological hallmark of branchioma is the identification of variable proportions of spindled cells, epithelial islands, and adipocytes [27]. The spindle cells are cytologically bland, and their appearance is either slender and delicate or plump and coarse [28]. The plump spindle cells tend to be arranged with fascicular, storiform, or lattice-like growth merged with the epithelial islands. Myoid differentiation, indicated by the presence of short fascicles of spindle cells with brightly eosinophilic cytoplasm, can be seen, particularly in circumferential arrangement around the epithelial elements [33]. Immunohistochemical studies have revealed coexpression of cytokeratin 5/6, p63, p40, and smooth muscle actin in the plump spindle cell component, suggesting myoepithelial differentiation [28, 29, 34]. The delicate spindle cells appear to be true stromal cells, as they are CD34 positive and cytokeratin negative [29, 35]. Myoid differentiation is highlighted by positive staining for myoglobin and muscle-specific actin (HHF35); however, those cells are negative for desmin [33]. In addition, variable degrees of androgen receptor, S100, and SOX10 reactivity has also been described, and PAX8 is consistently negative [29, 34, 36].

The epithelial islands are usually sharply demarcated from the spindle cells and most commonly consist of non-keratinizing squamoid cells with minimal atypia that are arranged in anastomosing cords of strands [27]. In some cases, glandular structures with ductal or acinar appearance can be identified. The glandular epithelium can show goblet cell morphology and contain ample eosinophilic cytoplasm [28, 34]. A rim of myoepithelial cells might be also present around the glandular epithelial islands [28, 34]. Multifocal areas of skin adnexal differentiation, including of sebaceous, eccrine, and apocrine elements, have been reported [36]. Cystic changes and focal keratinization in the form of keratohyalin structures have also been seen. The squamoid epithelial cells are characteristically positive for cytokeratin 5/6, p63, and p40; there may also be androgen receptor reactivity, particularly in the apocrine glandular elements. Similar to the spindle cells, all epithelial elements are negative for PAX8.

Three cases in which the epithelial element exhibited overt carcinomatous morphology have been reported [34, 37]. In these three cases, the malignant transformation affected the glandular epithelium and consisted of highly dysplastic cells, with apocrine-type eosinophilic cytoplasm showing cribriform architecture and Roman-ridge formation. The two cases described by Michal et al. resembled ductal carcinoma of the breast, and the one reported by Thompson et al. showed features of apocrine-type intraductal carcinoma [34, 37]. In all cases, the carcinoma was confined to the boundaries of the overall tumor, and the benign biological course of branchioma was not affected.

The third component of branchioma is mature adipose tissue. The amount varies from case to case, but it can represent up to 60% of the overall tumor area [29]. The distribution of the adipocytes is haphazard, and they tend to merge with the spindle cell elements in a pseudo-infiltrative or intermingling manner. The presence of mature lymphocytes was initially proposed as cardinal element of this tumor; however, this is not a conspicuous finding in all cases [27–29].

There is limited information regarding the molecular underpinnings of branchioma. Absence of PLAG1 rearrangements, as seen by fluorescence in-situ hybridization, has been reported in a series of 4 cases, and this feature further supports the notion that this tumor is not related to salivary gland mixed tumors (pleomorphic adenomas) [32]. Using a comprehensive targeted next-generation sequencing panel, Thompson et al. reported an HRAS c.181C>A (p.Gln61Lys) mutation in 1 case, but the other 2 cases their series lacked somatic driver mutations [34]. Interestingly, the HRAS alteration was identified in the tumor containing the apocrine-type intraductal carcinoma, which raises the possibility that this molecular finding might not be representative of all cellular components of branchioma.

Heterotopia-Associated Carcinoma

The concept of heterotopia-associated carcinomas was introduced in the fourth edition of the WHO Blue Book. This term refers to primary malignant epithelial neoplasms arising from histologically normal and congenitally displaced tissues, primarily ectopic thyroid and salivary tissue. Carcinomas arising from ectopic thyroid tissue occur along the path of the thyroglossal duct from the base tongue to the pyramidal lobe of the thyroid [38]. The 4th edition acknowledged the occurrence of ectopic papillary thyroid carcinoma in the lateral neck and in branchial cleft cysts; however, based on the embryological basis of the thyroid gland, follicular cells are not present in the lateral neck [39]. Consequently, the current WHO classification underlines that heterotopia-associated thyroid carcinoma is a midline phenomenon. In addition, this edition lists for the first-time diagnostic criteria for benign thyroid inclusion (Table 2).

Table 2.

Pathological features of benign thyroid inclusions listed by the WHO Classification of Tumors of the Head and Neck

Isolated, small focus or few in number of bland, simple follicles without cytologic atypia or architectural complexity Limited to one lymph node Capsular or immediately subcapsular location within lymph node Wedge-shaped arrangement, wider at capsule and narrow toward cortex Absence of psammomatous calcifications, fibrosis, or desmoplasia Absence of radiographic and/or pathologic abnormalities in the thyroid gland Absence of pathogenic mutations (e.g., BRAF V600E)

The incidence of carcinoma in thyroglossal duct cyst is about 3% [38]. Approximately 70% of cases occur in women, and the mean age at presentation is 40 years; however, a wide age range has been observed (9–83 years) [38]. Clinical presentation is usually indistinguishable from a regular thyroglossal duct cyst, except for rare incidences of fixed midline masses due to invasion. Therefore, over two-thirds thyroglossal duct cyst carcinomas are found incidentally after final pathologic examination of a presumed benign cyst. [40] Like eutopic thyroid cancer, the most common histological subtype is papillary thyroid carcinoma (Fig. 5), and the most frequent driver mutation is BRAFV600E. [38, 41].

Fig. 5.

Thyroglossal duct cyst carcinoma. A On scanning magnification, a multilocular structure containing papillary structures is observed, the lesion is surrounded by skeletal muscle and fibroconnective tissue; B the papillary structures are composed of tumor cells showing diagnostic nuclear features of papillary thyroid carcinoma; C adjacent ectopic benign thyroid follicles in close proximity to the hyoid bone; D residual thyroglossal duct cyst lined by benign squamous epithelium

Other histological subtypes of thyroglossal duct cyst malignancies are rarely reported, and squamous cell carcinoma appears to be the second most commonly documented type [40]. It should be noted that the thyroglossal duct apparatus does not participate in the migration of parafollicular C-cells which are derived from the ultimobranchial bodies that originate with the fourth pharyngeal pouches. Consequently, medullary thyroid carcinomas do not occur along the pathway of the thyroglossal duct [42].

Because of their midline location, pyramidal lobe carcinomas and thyroid carcinomas that metastasize to a level VI lymph node can be misinterpreted as thyroglossal duct cyst carcinomas. Correct diagnosis of thyroglossal duct cyst carcinomas can be aided by imaging and surgical findings coupled with pertinent histologic features, such as the presence of a respiratory and/or squamous epithelial-lined cyst, associated benign heterotopic thyroid tissue, and absence of lymph nodal architecture.

Heterotopia-associated salivary neoplasms are usually seen in periparotid and upper cervical nodes and are thought to arise from benign intranodal salivary inclusions. The intrauterine development of the parotid gland is a highly complex process that involves interaction of the ectoderm-derived oral epithelium with the underlying mesenchyme, which begins developing between 4 and 6 gestational weeks [43]. The encapsulation of the parotid gland by the superficial layer of the deep cervical fascia of the neck occurs after formation of the lymphatic networks and lymph nodes in the neck [43]. As a result, the parotid gland contains intraparenchymal lymph nodes and salivary epithelial elements can be entrapped within intra- and periglandular lymph nodes.

The morphologic features of heterotopia-associated salivary carcinomas are the same as tumors in eutopic locations; therefore, encountering an intranodal salivary carcinoma should raise the question of whether it is metastatic disease of an unknown primary. In such clinical scenarios, the main distinction relies on the absence of radiographic and/or pathologic abnormalities in any major or minor salivary glands, and the presence of a tumor within a lymph node that is detached from a salivary gland and contain benign intranodal salivary inclusions. In some cases, the diagnosis of a heterotopia-associated salivary carcinoma is only supported by long-term clinical follow-up. A primary intranodal salivary carcinoma should also be distinguished from an orthotopic salivary carcinoma with tumor-associated lymphoid proliferation (TALP); this determination is made based on the absence of nodal subcapsular sinus or hilum in TALP. In addition, a true lymph node contains extrafollicular reticulum cells which can be highlighted by a low molecular weight cytokeratin [44]. The identification of extrafollicular reticulum cells may help confirming the intranodal nature of salivary gland tumor.

The histological subtyping of heterotopia-associated salivary carcinomas follows the classification of salivary gland tumors presented in Chapter 4 of the fifth WHO Blue Blook. Based on case reports and limited series, mucoepidermoid and acinic cell carcinomas appear to be the most common histological subtypes [45, 46].

Recently, Rooper et al. described a subset of intraductal carcinomas (IDC) with intercalated duct-type differentiation within lymph nodes containing benign salivary epithelial inclusions [47]. These tumors did not completely fulfil the criteria for heterotopia-associated salivary carcinomas because the tumors involved intraparotid lymph nodes. However, this study expanded the list of salivary gland tumors that can arise from ectopic salivary gland tissue and reported a novel STRN-ALK fusion in IDC [47]. Further attention to these histological and molecular findings may lead to the identification of similar tumors in extraparotid lymph nodes. Of note, both the fourth and fifth editions of the WHO Blue Book focus on only salivary carcinomas; however, it is important to recognize that benign salivary gland tumors, particularly pleomorphic adenoma and Warthin tumor, also occur in ectopic locations [45, 46].

Carcinoma of Unknown Primary

The most common neoplastic lesions of the neck are cervical lymph node metastases that originate from a head and neck primary. Cases of proven or suspected cancer metastases to cervical nodes, particularly levels II/III, and no evident primary tumor site represent unique diagnostic challenges in this anatomic area. This clinical scenario is known as carcinoma of unknown primary (CUP), and it is estimated that 5% to 10% of primary head and neck cancers initially present as apparent CUP [48, 49]. In most cases a cancer in the oropharynx can be identified by means of multiple diagnostic modalities, including FDG-PET scans, panendoscopy, transoral laser microdissection, tonsillectomy, and/or base of tongue resection [48]. Therefore, the incidence true CUP is estimated to be less than 3% of cases initially diagnosed as CUP [48, 50]. Less commonly, primary cancers outside the head and neck region can present as CUP, and this phenomenon is discussed below in the section dedicated to metastasis.

Pathologists play an important role in the evaluation of cervical nodal metastases. Many cancers that tend to present as CUP have distinct morphological features and diagnostic biomarkers, which can help in identifying the primary site (Table 3). In addition, a cystic nodal metastasis in level II/III should raise a suspicion of an HPV-associated carcinoma arising in the tonsil or base of tongue base. The fifth edition of the WHO Blue Book recommends that high-risk (HR)-HPV testing should be routinely performed in the setting of CUP that exhibits squamous cell, undifferentiated, and neuroendocrine morphology (Fig. 6). As per CAP guidelines for HPV testing for head and neck carcinomas, p16 is recognized as a very sensitive surrogate marker of HR-HPV carcinogenesis [11]. However, the WHO Blue Book also recognizes the limitations of p16 immunohistochemistry for metastatic carcinomas located outside levels II/III and/or squamous cell carcinomas with keratinizing morphology. In these scenarios, additional specific HR-HPV testing using PCR-based assays or RNA in-situ hybridization should be performed to confirm HPV status [11, 51].

Table 3.

Carcinoma of unknown primary in the head and neck region: key diagnostic biomarkers

Suspected primary head and neck location	Diagnostic biomarkers
HPV-related oropharyngel squamous cell carcinoma	p16 and/or HR-HPV specific testing
EVB-related nasopharyngeal carcinoma	EBER
Cutaneous squamous cell carcinoma	UV-signature mutations
Merkle cell carcinoma	CK20, UV-signature mutations, MCPyV
HPV-related neuroendocrine carcinoma	p16 and/or HR-HPV specific testing
Laryngeal neuroendocrine tumor	Calcitonin (immunohistochemistry and serology)
Cribriform variant of polymorphous adenocarcinoma	PRKD1/2/3 gene rearrangement

Fig. 6.

Cystic metastatic HPV-associated carcinoma. A Nodal cervical metastasis of an oropharyngeal HPV-associated squamous cell carcinoma with extensive cystic change; B the metastatic tumor cells show non-keratinizing morphology and is cytomorphologically malignant; C tumor cells show diffuse and strong nuclear and cytoplasmic p16 positivity

Detection of Epstein-Barr virus encoded small RNA (EBER) in metastatic lymph nodes of patients with CUP strongly correlates with a nasopharynx origin [52]. If a nasopharyngeal carcinoma is histologically suspected and the metastatic carcinoma is EBER negative, p16 and/or HR-HPV specific testing is also recommended [53]. Conversely, if a non-keratinizing squamous cell carcinoma is negative for p16 and/or specific HR-HPV testing, performing EBER is advised.

Head and neck cutaneous squamous cell carcinoma is often a underrecognized source of CUP, and distinguishing it from a metastatic HPV-associated carcinoma can be challenging because 20% of cutaneous head and neck squamous cell carcinomas exhibit p16 positivity unrelated to HR-HPV-driven carcinogenesis [54]. Ultraviolet (UV) radiation exposure is the most important risk factor for the development of cutaneous squamous cell carcinoma, and it imprints a specific pattern of DNA damage. These patterns are known as UV signature mutations and consist of C → T substitutions at dipyrimidine sites in ≥ 60% of all mutations, with ≥ 5% CC → TT [55]. The identification of UV signature mutations in CUP indicates a cutaneous primary [56]. Of note, the UV signature mutations are not only restricted to cutaneous squamous cell carcinoma; they are also present in Merkel cell polyomavirus (MCPyV) negative MCC, which can also present as CUP [57].

In rare occasions, a cervical lymph metastasis may be the first presentation of a neuroendocrine neoplasm; adequate tumor classification following the WHO/IARC unified terminology framework for neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs) can help with selecting the most appropriate diagnostic biomarkers [58]. The supraglottic larynx can be the primary site for a NET with immunoreactivity for calcitonin but normal serum levels; only rare cases of laryngeal NETs have been reported to have elevated serum calcitonin levels. A metastatic NEC could originate from the oropharynx, and positive HR-HPV testing most likely indicates a tonsillar and/or base of tongue primary site [59]. Lastly, the cribriform variant of polymorphous adenocarcinoma usually presents with metastases in the neck lymph nodes that can precede the identification of the primary tumor [60].

Metastasis to the Head and Neck Region

Following the trend established by of other WHO Blue Books of the fifth edition, the classification of head and neck tumors introduces a chapter dedicated to metastatic diseases originating from organ systems in the thorax, abdomen, and pelvis. Metastatic dissemination can occur through hematogenous spread using pulmonary circulation via the paravertebral plexus of Batson or through lymphatic networks, including the thoracic duct [61]. The latter mechanism leads to metastatic carcinoma involving left supraclavicular lymph nodes, a clinical observation known as Virchow node or Troisier sign [62]. Virtually any malignancy originating bellow the clavicle can be the source of a metastasis to the head and neck region, and affected compartment can provide clues on the possible primary source (Table 4). Clinically, metastatic disease can be identified during clinical and radiological staging of a known malignancy, manifest during short- or long-term surveillance, or be the first presentation a tumor (i.e., non-head and neck CUP).

Table 4.

Common locations and primary origins of metastases to head and neck region

Head and neck compartment	Common localization	Common primary non-head and neck origin
Neck	Lower neck lymph node levels Left supraclavicular lymph node (Virchow node or Troisier sign)	Breast and lung Pelvic/intra-abdominal (Virchow node or Troisier sign)
Oral cavity	Tooth-bearing gingiva >  > tongue	Women: Breast, kidney (preferentially tongue) and skin Men: Lung (preferentially tongue)
Major salivary glands	Parotid gland Intra- and periglandular lymph nodes	Lung, breast, kidney, stomach, and prostate
Oroharynx and nasopharynx	Base of tongue Palatine tonsil Posterior nasopharyngeal wall Fossa of Rosenmüller	Lung, gastrointestinal tract, renal, breast
Sinonasal tract	Maxillary > sphenoid > ethmoid > frontal sinuses	Lung, breast, kidney
Ear	Petrous apex	Breast and müllerian tract (women), lung and prostate carcinoma (men), kidney, melanoma
Larynx	Mucosal involvement (transglottic)	Melanoma, kidney, lung, breast
Maxillofacial bones	Mandible (molar area) ≫ maxilla	Breast ≫ lung, colorectal, renal cancers

As a rule, when confronted with a malignancy in any head and neck site that poses diagnostic challenges, distant metastatic disease should be considered. The histological, immunohistochemical, and molecular profiling of metastatic tumors mirrors the findings of the primary lesions, and extensive ancillary testing might be required. Importantly, clinical history, multidisciplinary discussion, imaging findings, and comparison with any available prior diagnostic material are key in rendering the correct diagnosis. Pathologists should be also aware that some primary head and neck tumors are known to have overlapping morphological and immunohistochemical features with tumors originating from other organ systems (Table 5).

Table 5.

Metastases to head and neck with morphological overlap with primary head and neck malignancies

Metastasis	Primary
Breast carcinoma	Salivary duct carcinoma
Lung adenocarcinoma	Papillary thyroid carcinoma
Clear cell renal cell carcinoma	Clear cell carcinoma of salivary glands Renal cell-like adenocarcinoma
Gastrointestinal adenocarcinoma	Intestinal-type sinonasal adenocarcinoma

Melanocytic Tumors

In keeping with other Blue Books of the edition, the WHO Classification of Head and Neck Tumors combines the discussions on mucosal melanomas into 1 chapter. These rare mucosal-based tumors can arise in the sinonasal tract, oral cavity, or larynx, with slight variability in their clinicopathological features; importantly, at each anatomic site, the differential diagnoses that need to be considered are different (Table 6). Immunohistochemistry is virtually always used to diagnose mucosal melanomas, particularly in amelanotic lesions. However, as highlighted by the WHO Blue Book, mucosal melanomas have variable sensitivity for S100 protein and melanocytic markers, including HMB45, tyrosinase, melan-A, MITF, and SOX10, depending on the morphological tumor type and location [63, 64]. Compared to spindle cell melanomas, tumors with epithelioid morphology show more consistent expression for these markers (Fig. 7) [63, 64]. Among mucosal melanomas from the sinonasal tract, there is variable expression for S100 protein, which limits its use as a screening marker; SOX10 shows more consistent and robust positivity [64, 65].

Table 6.

Head and neck mucosal melanoma

	Sinonasal tract	Oral cavity	Larynx
Localization	Nasal septum, turbinate, maxillary sinus	Maxillary gingiva and hard palate	Supraglottic larynx
Frequency	4% of sinonasal tumors	0.26% of oral malignancies	Extremely rare
Macroscopic appearance	Polypoid, exophytic nodules	Pigmented flat or nodular lesions	Polypoid, exophytic nodules
Peak incidence	7th decade	6th decade	6th–7th decade
Differential diagnosis based on anatomic location	Olfactory neuroblastoma, Ewing sarcoma, rhabdomyosarcoma, sinonasal undifferentiated carcinoma, poorly differentiated squamous cell carcinoma, NUT carcinoma, SMARCB1-deficient carcinoma, neuroendocrine carcinomas, lymphoma, extramedullary plasmacytoma	Amalgam tattoos Oral nevi Melanoacanthoma Smoker’s melanosis Spindle cell squamous cell carcinoma	Poorly differentiated squamous cell carcinoma, spindle cell squamous cell carcinoma Neuroendocrine tumors and carcinomas, laryngeal paraganglioma, lymphoma, extramedullary plasmacytoma

Fig. 7.

Sinonasal mucosal melanoma. A Subepithelial malignant neoplastic proliferation containing melanin pigment; B tumor cells are show epithelioid morphology with round to ovel nuclei with prominent nucleoli; C. tumor cells are diffusely positive for SOX10

From a molecular and clinical perspective, mucosal melanomas are markedly different from cutaneous and uveal melanomas. Overall, mucosal melanomas have significantly lower mutational burden, lower frequency of BRAFV600E mutations, a lack of UV signature mutations, a higher KIT mutation rate, and poorer responses to immunotherapy [66]. Since the fourth edition of the WHO Blue Book, the molecular landscape of mucosal melanomas across different anatomic sites has expanded to include SF3B1 R625 mutations and co-existing NF1 and KIT mutations [67]. Relative to the head and neck mucosal melanomas, the mutational profile is similar to upper gastrointestinal tract melanomas, and as a group, BRAF, NRAS, NF1, KIT and SF3B1 mutations are documented in 6%, 12%, 10%, 15% and 6% of cases, respectively [66].

SF3B1 encodes the subunit 1 of the splicing factor 3b protein complex and has a major role in RNA splicing, during which precursor messenger RNA (pre-mRNA) transcripts are transformed into a mature messenger RNA (mRNA) by removal of noncoding introns [68]. SF3B1 mutations have been identified multiple tumor types and lead to dysregulated mRNA splicing [68]. These alterations are of great clinical interest, as a phase 1 clinical trial using an oral spliceosomal inhibitor to treat advanced myeloid malignancies (NCT02841540) is currently recruiting patients and might lead to a treatment option for patients with SF3B1-mutant mucosal melanoma.

Conclusion

There are a couple new entries in Chapter 11 of fifth edition of the WHO Classification Head and Neck Tumors. Ectopic hamartomatous thymoma has been incorporated for the first time under the name of (biphenotypic) branchioma. Lymphoepithelial cyst has also been newly added, and time will prove the validity of its inclusion. The WHO endorses the recommendations of CAP regarding HPV testing for carcinomas of unknown primary and UV signature mutations are presented as an ancillary diagnostic tool for metastatic cutaneous carcinomas. The new Chapter 15 is dedicated to the discussion of metastases to the head and neck region, and it provides a list of selective and targeted immunohistochemical markers useful in supporting the diagnosis of non-head and neck primary origin. The combined discussion of mucosal melanomas in Chapter 10 highlights the emerging molecular landscape of these tumors, which is leading to the recognition of potential molecular therapeutic targets.

Author Contributions

Conceptualization, literature search, data analysis, writing, review and editing by JHP.

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Declarations

Conflict of interest

The author declare that they have no conflict of interest as it relates to this research project.

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This review article did not involve the use of human or animal subjects and has complied with the ethical standards as outlined by the publisher.

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