Molecular pathology in diagnosis and prognostication of head and neck tumors

Classification of head and neck tumors has evolved in recent decades including a widespread application of molecular testing in tumors of the salivary glands, sinonasal tract, oropharynx, nasopharynx, and soft tissue. Availability of new molecular techniques allowed for the definition of multiple novel tumor types unique to head and neck sites. Moreover, the expanding spectrum of immunohistochemical markers facilitates a rapid identification of diagnostic molecular abnormalities. As such, it is currently possible for head and neck pathologists to benefit from a molecularly defined classifications, while making diagnoses that are still based largely on histopathology and immunohistochemistry. This review highlights some principal molecular alterations in head and neck neoplasms presently available to assist pathologists in the practice of diagnosis, prognostication and prediction of response to treatment.


Introduction
Classification of head and neck tumors has expanded dramatically in recent decades with identification of many new entities and widespread application of molecular testing in tumors of the salivary glands, sinonasal tract, oropharynx, nasopharynx, and soft tissue of the head and neck.Molecular testing has disclosed pathogenetic processes of wellestablished but previously enigmatic entities, and clarified the relationships between various neoplasms.The current 5th edition of the WHO Classification of Head and Neck Tumours relies heavily on molecular data to support the inclusion of several new tumor entities and their subtypes, and to provide prognostic and pathogenetic information [1].Furthermore, the description of molecular alterations in various subgroups of tumors and the application of molecular testing in large case cohorts in a research setting has substantially clarified the boundaries of many existing histologic entities and allowed for the identification of specific morphologic and immunohistochemical features of new tumor types.On the other hand, it must be emphasized that this molecular revolution in head and neck pathology has not made pathologists entirely dependent on molecular testing in making their diagnoses.An expanding spectrum of immunohistochemical markers based on new molecular information has improved diagnostic options using immunohistochemistry, and such markers also facilitate a rapid identification of useful diagnostic molecular tests.Thus, head and neck pathologists currently benefit from a molecularly defined classification, while making diagnoses that are still based largely on histopathology and immunohistochemistry.
In recent years, considerable progress in salivary gland and sinonasal tumor taxonomy has taken place with the discovery of tumor type-specific fusion oncogenes generated by chromosomal translocations.This review covers molecular alterations important in head and neck neoplasms from a practical viewpoint in diagnosis, prognostication and prediction of response to treatment.

Salivary gland neoplasms
Salivary gland tumors represent a challenging field in diagnostic head and neck pathology.These rare tumors exhibit a diverse array of morphological features, comprising almost 40 subtypes in the current World Health Organization (WHO) Classification of Head and Neck Tumours [1].Traditionally, salivary neoplasms are diagnosed on morphological grounds, with the help of ancillary stains and immunohistochemistry. Several salivary gland neoplasms are characterized by recurrent genomic alterations, particularly gene fusions.These include fusions of the ETV6 gene in secretory carcinoma (SC) [2][3][4], MYB/MYBL1::NFIB in adenoid cystic carcinoma (AdCC) [5], CRTC1/CRTC3::MAML2 in mucoepidermoid carcinoma (MEC) [6,7], and EWSR1::ATF1 in hyalinizing clear cell carcinoma [8] as the most frequent and best characterized gene fusions (Table 1).

Molecular alterations with prognostic and predictive significance
Salivary gland secretory carcinoma (SC), previously called mammary analogue SC, is a mostly low-grade malignancy characterized by well-defined morphology and an immunohistochemical (Fig. 1A-D) and genetic profile identical to SC of the breast [2].Translocation t(12;15)(p13;q25) resulting in the ETV6::NTRK3 gene fusion is characteristic for SC, along with S100 protein and mammaglobin immunopositivity.The spectrum of genetic alterations in rare cases of SC continues to evolve including cases with ETV6::RET [3], ETV6::MET [9], and VIM::RET [10] fusions.Other rare alterations in low-grade SC include CTNNB1::ALK fusion [11] and dual fusions of ETV6::NTRK3 and ETV6:: MAML3 in the same tumor [12].All these rearrangements can be used as diagnostically useful molecular markers in establishing the diagnosis of SC.Immunohistochemical positivity for S100, SOX10 and mammaglobin is a practical way to diagnose SC, and molecular testing may not be needed in most cases.
Salivary mucoepidermoid carcinoma (MEC) is a malignant salivary gland neoplasm characterized by mucous, intermediate and epidermoid (squamoid) tumor cells forming cystic and solid growth patterns, and usually associated with MAML2 gene rearrangement.Most MECs harbor a tumor type-specific translocation at t(11;19)(q21;p13) resulting in CRTC1::MAML2 fusion gene [6,7,17].Rare cases display a t(11;15)(q21;q26) translocation with CRTC3::MAML2 fusion [18] (Fig. 2A-C) or a highly unusual t(6;22)(p21;q12) translocation with EWSR1::POU5F1 fusion [19].The CRTC1::MAML2 fusion is seen in most low-and intermediate-grade and some high-grade tumors.CRTC1/3::MAML2 fusions were originally considered as a biomarker for favorable overall survival in patients with salivary gland MEC [7].More recent studies, however, have challenged these conclusions and current grading systems have not been fully satisfying in predicting the aggressiveness of MECs.A positive FISH-result for MAML2-rearrangement can be highly useful in confirming the diagnosis of MEC in difficult cases.Further evidence may be gained from NGS and PCR testing.However, negative FISH results are inconclusive, and cannot be used in differential diagnosis.The immunohistochemical profile of MEC includes positivity for p63, p40, and may be useful in differential diagnosis from other salivary gland tumors.In one recent study, KMT2A gene rearrangements in MECs identified aggressive tumor behavior regardless of histologic grade obtained with conventional grading systems [20].Furthermore, these results may suggest the possibility of treating KMT2A-rearranged MECs using azacytidine and venetoclax, as these treatments have shown promising outcomes in the treatment of KMT2Arearranged leukemias [21].
Adenoid cystic carcinoma (AdCC) is an invasive carcinoma composed of epithelial and myoepithelial neoplastic cells arranged in tubular, cribriform, and solid patterns associated with an eosinophilic extracellular matrix and reduplicated basement membrane materials, often associated with gene fusions involving the MYB, MYBL1 and NFIB genes.The genomic hallmarks of AdCC are t(6;9) or t(8;9) translocations, resulting in MYB::NFIB and MYBL1::NFIB fusions, respectively [5,22] (Fig. 3A-D).The former alteration is found in > 80% of cases and the latter in approximately 5% of cases [5].MYB/MYBL1 activation due to gene fusion or other mechanisms is a key event in pathogenesis of AdCC [22].Losses of 1p, 6q, and 15q are associated with highgrade tumours, and loss of 14q is seen exclusively in lowgrade tumors [23,24].Next-generation sequencing identified mutations with mostly low level of recurrence in genes of the FGF/IGF/PI3K, chromatin remodelling, and NOTCH signaling pathways [25,26].Use of FISH techniques for MYB, MYBL1 or NFIB-rearrangments can be very useful for specific identification of AdCC.Immunohistochemical antibodies to altered MYB can also be used.
Traditional therapeutic agents for patients with advanced, recurrent, or metastatic AdCC have demonstrated poor efficacy in prolonging survival.The presence of NOTCH alterations in AdCC associate with poor survival.Patients with these aberrations might potentially benefit from anti-NOTCH drugs, such as bronticuzumab [27].
Salivary duct carcinoma (SDC) is an aggressive malignancy resembling mammary ductal carcinoma, most typically with an apocrine phenotype.The most frequent genetic alterations are mutations in TP53 (55%), HRAS (23%), PIK3CA (23%), and an amplification of HER2/ neu (35%) [28].Due to low recurrency of these mutations,  PLAG1 or HMGA2 rearrangements due to a pre-existing pleomorphic adenoma (PA) can be identified in about half of SDCs [29,30].A small subset of SDCs harbors a rearrangement of ALK [31].Given the therapeutic relevance of ALK fusions, inclusion of ALK immunohistochemistry in any atypical-looking or androgen receptor poor SDC and high-grade adenocarcinoma, not otherwise specified is recommended.Because systemic therapies targeting androgen receptor, HER2/neu amplification, phosphatidylinositol 3 kinase (PI3K) pathway, including mutations of PIK3CA and BRAF p.V600E, and loss of phosphatase and tensin homolog (PTEN) are presently under study, molecular testing for them may become warranted [32][33][34].

Molecular alterations with differential diagnostic significance
Hyalinizing clear cell carcinoma (HCCC) is a carcinoma composed of clear and eosinophilic cells in a variably hyalinized stroma, usually associated with EWSR1 rearrangement.
Although the finding of infiltrative nests of low-grade clear tumor cells within a hyalinized or cellular fibrous stroma is quite characteristic of HCCC, some cases may have also abundant mucocytes and/or clear cell differentiation, or the hyalinization is minimal or absent.Differential diagnosis in such cases includes a wide spectrum of salivary gland tumors, namely clear cell mucoepidermoid carcinoma, epithelial-myoepithelial carcinoma, myoepithelial carcinoma, oncocytoma, myoepithelioma, and even metastatic kidney clear cell carcinoma, and the distinction of these benefits from FISH studies [36] on EWSR1-rearrangment.Conventional immunohistochemistry may also be helpful, but not always conclusive.Epithelial-myoepithelial carcinoma (EMC) is a salivary gland malignancy characterized by biphasic tubular structures and usually a multinodular growth pattern.The histological hallmark of EMC is a biphasic arrangement of inner (luminal) eosinophilic ductal epithelial cells and outer (abluminal) cells which are often clear myoepithelial cells.There are, however, several histologic variants with cribriform, basaloid, sebaceous, apocrine/oncocytic, or double-clear appearances, or with squamous differentiation resulting in differential diagnostic challenges.The salivary gland tumor entities to be differentiated include those with biphasic or/and clear cell morphology, such as adenoid cystic carcinoma, basal cell adenocarcinoma, pleomorphic adenoma, myoepithelial carcinoma, and clear cell carcinoma [37].Because clear myoepithelial cells can be seen in many benign and malignant salivary gland tumors, the differential diagnosis of epithelial-myoepithelial carcinoma is challenging.Testing for HRAS mutations is useful when discriminating EMC from its mimics [38].Immunohistochemistry is often not conclusive.
Polymorphous adenocarcinoma (PAC) (previously known as polymorphous low-grade adenocarcinoma) is a malignant epithelial tumor characterized by cytological uniformity, morphological diversity, and an infiltrative growth pattern.It is predominantly seen in minor salivary glands.Cribriform adenocarcinoma (CAC) was initially reported at the base of the tongue [39] and later in other minor salivary gland locations [40].CAC is characterized by a multinodular growth pattern separated by fibrous septa, a relatively uniform solid, cribriform and microcystic architecture, and optically clear nuclei.Glomeruloid and papillary structures, peripheral palisading and clefting may be observed.Although CAC and classic PAC have molecular alterations affecting the same PRKD gene family, there are notable differences.PACs harbor recurrent PRKD1 E710D hotspot mutations in > 70% of cases, whereas 80% of CACs display PRKD1/2/3 fusions with partner genes including ARID1A, DDX3X or STRN3 [41,42].Histologically, fusion-positive tumors show papillary growth in a high percentage of cases, while arrangement in single cell filing is seen in a low percentage.CACs display a propensity for base of tongue location and frequent (50%) lymph node metastasis compared with the hot spot mutation-related tumors which have a low risk for nodal metastasis.Compared with classic PAC, CAC has a high propensity for location in the base of the tongue and a higher risk for lymph node metastasis [43].Genetic analysis of PRKD genes appears to be useful in characterizing this spectrum of tumors, and identifying those tumors with a high risk for nodal metastasis.Immunohistochemistry may not be conclusive with this regard.Pleomorphic adenoma (PA) is a benign tumor characterized by cytomorphological and architectural diversity with an admixture of ductal and myoepithelial cells usually embedded in a chondromyxoid or fibrous stroma.PAs harbour recurrent translocations or intrachromosomal rearrangements resulting in gene fusions involving PLAG1 on 8q12 (> 50%) or HMGA2 on 12q14.3(10-15%) [44,45].Antibodies to PLAG1 [46] and HMGA2 [47], respectively, are emerging as practical, sensitive and specific immunohistochemical markers for PA.
Diagnosis of PA is straightforward in most cases but sometimes PA is difficult to differentiate from low-grade epithelial-myoepithelial carcinoma.Molecular testing for PLAG1/HMGA2 rearrangement versus HRAS mutation can be very useful.

Sinonasal neoplasms
Sinonasal tract comprising the nasal cavity, paranasal sinuses and the skull base is a region characterized by a broad spectrum of tumors that exhibit a significant diversity of molecularly defined entities (Table 2).The recent WHO Classification of Head and Neck Tumors includes new entities in which molecular genetics has an important diagnostic role, including HPV-related multiphenotypic sinonasal carcinoma and SWI/SNF-deficient sinonasal carcinoma and adenocarcinoma [1], and a subset of emerging and developing entities including IDH-mutated malignancies in the category of sinonasal undifferentiated carcinoma.Molecular genetics also plays a diagnostic role in the context of hereditary syndromes that can be manifested in the sinonasal tract.
Non-keratinizing squamous cell carcinoma (NKSCC) with DEK::AFF2 gene fusion.Sinonasal NKSCC is in 36-58% driven by transcriptionally active HPV, most commonly type 16 [1].DEK::AFF2 carcinoma is currently classified as an emerging entity under NKSCC category, localized especially in the sinonasal tract but also few cases were reported in the middle ear [49,50].Despite bland looking morphology this tumor behaves in and aggressive fashion with high-risk of local recurrence, nodal metastatic dissemination and distant spread.FISH testing for DEK-or AFF2 rearrangements is essential for diagnosis.DEK::AFF2 carcinoma has an excellent response to immune checkpoint inhibitors (ICI) -anti-PD-L1 [49].
NUT Carcinoma is a highly aggressive, mostly lethal malignancy with a monotonous poorly differentiated morphology.NUT carcinoma is genetically characterized by rearrangement of NUTM1 gene on 15q14 [51].The NUT gene is physiologically expressed in mature spermatogonia.The most common fusion partners of NUT are genes involved in transcription and chromosome regulation belonging to the BET family (BRD2, BRD3, BRD4 and BRDT) [52].In 75% of cases the fusion partner of NUTM1 is the BRD4 gene (in 19p13.1),and in 15% the BRD3 gene (in 9q34.2) [53,54].FISH techniques or other molecular testing may be used to demonstrate NUTM1-and the various BRD-rearrangements.However, the monoclonal NUT antibody is highly specific for NUT carcinoma [55], and offers a simple and reliable way to diagnose this malignancy.
There is no known effective treatment for NUT carcinoma which has a median survival of 6.7 months [58].The first targeted drugs for NUT carcinoma were histone deacetylase inhibitors (HDACi) (vorinostat) and BET inhibitors (BETi) [59].
These tumors are usually poorly differentiated or undifferentiated malignancies with a highly aggressive clinical course and poor outcome.
The mortality of SMARCA4-deficient sinonasal carcinomas is higher than in other tumors of this family [61,62].SWI/SNF mutations are involved in many cancerrelated pathways mostly related to epigenetic alterations (Fig. 6A-C).SWI/SNF complex deficient malignancies can be preferentially identified by immunohistochemical antibodies to SMARCB1 (INI1) and SMARCA4 (BRG1) which are sensitive diagnostic tools [63].The therapeutic option rests in local control of the disease with polychemotherapy and radiotherapy [61,62].
Recent findings indicate a promising role for immunomodulators and immune checkpoint inhibitors as potential drugs in patients with SWI/SNF related malignancies [60,64].Tumor cells with loss of SMARCB1 demonstrate a constitutive EZH2 activation, and EZH2 inhibitors may modulate tumor immunogenicity and anti-tumor immune response [60,64,65].
Sinonasal undifferentiated carcinoma (SNUC) is a high-grade epithelial neoplasm without signs of cell differentiation, and the diagnosis is made only by exclusion of other sinonasal and non-sinonasal malignancies.Many undifferentiated epithelial neoplasms in previous WHO classifications were included under this term until advances in molecular pathology allowed for their identification as proper entities.Recently, isocitrate dehydrogenase 1 or 2 (IDH1/2) mutations were identified in a subset of SNUC [66,67] including three main hotspot mutations of IDH1 R132, IDH2 R140 and IDH2 R172 [68].Monoclonal or multi-specific antibodies for immunohistochemical detection of IDH1/2 mutations represent a cheaper alternative to molecular genetic testing for these mutations.However, immunohistochemistry lacks the ability to detect the full spectrum of IDH1/2 mutations [69].
SNUCs are aggressive diseases with poor outcome.The standard therapeutical approach is a combination of surgery, chemotherapy and radiation [70].Tumors with IDH2-mutations show better outcome than other SNUCs [70].IDH mutations provide alternative therapeutic options including selective small molecule inhibitors (e.g.Enasidenib for IDH2 mutations and Ivosidenib for IDH1 mutations).
HPV-associated multiphenotypic sinonasal carcinoma (HMSC) is an epithelial tumor almost exclusively localized in sinonasal tract and harboring high-risk HPV [71].The most common serovariety is type 33.P16 immunohistochemistry can be used to identify the HPV-association.HMSC is histologically very pleomorphic and may mimic various salivary and non-salivary tumor types.The histological appearance of HMSC is usually high-grade and associated with destructive growth and propensity for local recurrence.Despite the aggressive appearance, HMSC has low metastatic potential and little tendency to lethal behavior [72].

Molecular alterations with differential diagnostic significance
The head and neck can be host to a wide range of soft tissue neoplasms with molecular findings, but a discussion of all these entities is beyond the scope of this review.We have included a selection of soft tissue tumors, often of recent molecular definition, that harbor diagnostically obviously useful molecular alterations.All discussed entities are listed in Table 3.
BSS are usually low-grade tumors with frequent local recurrence, sometimes many years after diagnosis [77].Three cases of BSS with high-grade transformation have been published [78][79][80], one of which developed into rhabdomyosarcoma (RMS).Molecular testing of BSS and RMS showed similar gene fusions including PAX3::FOXO1 and PAX3::NCOA1 [74].
Nasal chondromesenchymal hamartoma (NCMH) is a pediatric benign mesenchymal sinonasal tumor which in a subset of cases is associated with DICER1 syndrome [81].NCMH is usually an indolent tumor but when syndromic, patients have an increased risk for pleuropulmonary  [82,83].IMT is driven by constitutive activity of ALK or other receptor tyrosine kinase.Immunostaining for ALK-1 serves as a valuable screening tool, and ALK rearrangement can be confirmed with FISH techniques.Rare cases of IMT with fusions of other tyrosine kinase genes suggest that in cases of ALK-1 immunonegativity targeted RNA-sequencing would be a more suitable test.
Ectomesenchymal chondromyxoid tumor (ECT) (aka RREB1::MRTFB-rearranged neoplasm) is a tumor of uncertain malignant potential located predominantly in the tongue and rarely arising in extraglossal locations [84,85].The immunoprofile is non-specific, and therefore molecular testing is the preferred method when diagnosing ECT.In most cases it is characterized by a RREB1::MRTFB fusion, while EWSR1 gene rearrangement is seen in a small subset of cases [86,87].These tumors are genetically and histologically linked to soft tissue myoepithelial tumors [86] and may be mistaken for other mesenchymal tumors with dominant spindle cell morphology [88].ECT usually follows benign course with no metastasis.Surgery is curative in most cases, even though local recurrences may occur [84,85].
GLI1-altered soft tissue neoplasms are tumors of uncertain histogenesis, epithelioid morphology and non-specific immunoprofile, presenting in the head and neck in 40% of cases.In two thirds of cases these tumors harbor GLI1 fusions including ACTB::GLI1, PTCH1::GLI1, MALAT1::GLI1, and DERA::GLI1, while the rest harbor GLI1 amplification [1,89,90].The various fusion partners to GLI1 are best determined by targeted RNA-sequencing but GLI1 amplification can also be detected by FISH.Awareness of a potential co-amplification of neighboring genes (CDK4, MDM2, DDIT3, etc.) on chromosome 12 detectable by FISH has to be taken into account [91].GLI1 immunostaining shows high specificity and good sensitivity for GLI1-rearranged mesenchymal tumors.However, the sensitivity is unsatisfactory in plexiform fibromyxoma [92].Biologic behavior varies from indolent tumors to metastasizing malignancies.Local recurrence or distant spread may appear in approximately 20% of cases [90,93].Potential targeted therapeutic options in GLI1-altered neoplasms are sonic hedgehod (Shh) signaling pathway inhibitors [94].
TFCP2-rearranged RMS (TFCP2-RMS) are spindle cell/sclerosing and very aggressive mesenchymal tumors with rhabdomyoblastic differentiation and EWSR1/ FUS::TFCP2 rearrangements [100].In a subset of cases, hemizygous deletion or amplification of the ALK gene are described [101].TFCP2-RMS tumors are predominantly localized in the jaws and the skull of young adults.The tumors have a rapid clinical course with a poor prognosis of 3-year overall survival at 28% [102].Treatment options include surgery.chemotherapy and radiotherapy.The combination of radiation and ALK inhibitors has shown partial response in anecdotal cases [103,104].
Adamantinoma-like Ewing sarcoma (ALES) is a controversial variant of Ewing sarcoma (ES) defined by the presence of t (11;22) and EWSR1::FLI1 fusion [105].It is speculated whether they represent an epithelial or mesenchymal neoplasm, as they commonly express epithelial markers (pancytokeratins and p63/p40) as well as ES-related markers (CD99 and NKX2.2) [106].Many tumors are treated with surgery and adjuvant polychemotherapy according to ESspecific protocols.
NTRK-and other kinase gene-rearranged spindle cell neoplasms encompass a wide morphological spectrum ranging from benign-appearing to high-grade tumors, consisting of spindled cells often immunopositive for S100 protein and CD34, and resembling infantile or adult-type fibrosarcoma, malignant peripheral nerve sheath tumor, or lipofibromatosis-like neural tumor.Superficial and deep soft tissues including salivary glands, as well as the skeletal component of the head and neck, are some of the more common locations of these tumors [107,108].
While ETV6::NTRK3 is a well-established genetic driver of infantile fibrosarcoma, other fusions of NTRK1/3 and various other kinase genes including MET, RET, RAF1, or BRAF have been implicated in the pathogenesis of these tumors.Most cases are only locally aggressive and distant metastasis is an uncommon event in high-grade cases [108].PanTrk immunohistochemistry seems to be a useful screening tool in NTRK1/3 rearranged cases with an overall sensitivity of 88% [109].FISH detection of ETV6::NTRK3 is sufficient to confirm the diagnosis of infantile fibrosarcoma.However, given the number of possible kinase genes and their fusion partners when other tumor entities come into question, targeted RNA-sequencing is the method of choice [108].Importantly, small inhibitory molecules targeting NTRK and RET have been used in the treatment of locally advanced inoperable tumors or cases with metastatic spread.
EWSR1/FUS::POU2AF3(COLCA2) sarcomas are newly recognized aggressive neoplasms with tendency to both local recurrence and metastatic spread despite multimodal treatment.They affect adult patients and commonly arise in the head and neck, particularly within the sinonasal tract.Their morphological spectrum of these tumors is wide from spindle cells, through biphasic tumors with spindled and round cells with features of neuroendocrine differentiation, to purely round cell tumors with high nuclear grade [110][111][112].Approximately half of the cases display pancytokeratin positivity [110][111][112], possibly leading to diagnostic confusion with synovial sarcoma.As EWSR1 fusions with various partners are frequent in soft tissue tumors, FISH analysis of an EWSR1 break is not a sufficient diagnostic test.Instead, targeted RNA-sequencing is advised to render the correct diagnosis.

HPV-And EBV-Associated tumors of the nasopharynx and the oropharynx
The expression of late viral genes plays a role in the pathogenesis of epithelial and lymphoproliferative neoplasms of the oropharyngeal and nasopharyngeal areas.Only a considerably small subset of patients who acquired the infection during their lifetime develop any related tumor, highlighting the major influence of other genetic and environmental factors in their pathogenesis.
Nasopharyngeal carcinoma (NPC) is a rare neoplasm with high incidence in high-risk (endemic) populations including ethnic Chinese (southern China and Singapore) and Inuits of Alaska, Canada and Greenland.Low-risk (nonendemic) populations with rare occurrence of NPC include Europeans and Americans of Caucasian ethnicity [1].In most cases of non-keratinizing NPC in high-risk populations, EBV infection is an early pathogenetic event [113].Other important pathogenetic influences include genetic and environmental factors [114].In low-risk populations, fewer cases are EBV positive, while high-risk HPV positive and virus-negative cases, particularly in keratinizing NPC are seen [115,116].In-situ hybridization with EBV-encoded small RNAs (EBER) or RNA-ISH for high-risk HPV can be used to detect the viral load in tumor cells.Immunohistochemistry for LMP-1 has low sensitivity and is not recommended.Serum EBV DNA and antibody levels have prognostic value, and patients with high viral load fare worse than those with lower load or absence of viral DNA [114].In EBV-and HPV-positive cases patients have shown better outcome than in virus-negative cases [115].
HPV-associated squamous cell carcinoma of the oropharynx has an increasing incidence especially in developed countries.In North America and Northern Europe it is more prevalent now than the HPV-independent oropharyngeal squamous cell carcinoma [117].High-risk HPV16 is responsible for most cases of HPV-associated carcinoma [118].The patients are generally younger, have a higher socioeconomic status and have a better prognosis (5-year overall survival of 80%) than patients with HPVindependent carcinoma [1,119,120].However, similar to HPV-independent cases, patients with HPV-associated squamous cell carcinoma are often active or past smokers [119].The HPV-associated carcinomas most commonly originate from the squamous epithelium of tonsillar crypts and lack keratinization [121].Histologic grading is not recommended as it does not carry significant prognostic value.In addition, the HPV-associated neuroendocrine carcinoma is a distinct aggressive tumor type that must be differentiated from squamous cell carcinoma.This can be facilitated by positive immunohistochemical staining for neuroendocrine markers such as synaptophysin, chromogranin and INSM1.Immunohistochemistry for the surrogate marker p16 is sufficient to detect HPV infection in most cases [122].Strong diffuse positivity in tumor cell cytoplasm and nuclei must be present in more than 70% of cells to diagnose the lesion as positive.In problematic instances (equivocal p16 positivity, discrepancy between morphology and immunoprofile), PCR genotyping or in-situ hybridization methods can be employed to confirm the presence of high-risk HPV in the tumor.
Extranodal NK/T-cell lymphoma, nasal type is an aggressive tumor that infiltrates and destroys the nasal cavity, the nasopharynx, the oropharynx and structures of the oral cavity.It belongs to EBV-positive lymphoproliferations, and insitu hybridization for EBER is positive in most viable tumor cells [1].While a 5-year overall survival can be achieved using chemoradiotherapy in a majority of patients, the detection of persistent post-treatment serum EBV DNA suggests a worse prognosis [123].
EBV-associated smooth muscle tumor and EBV-positive mucocutaneous ulcer are two indolent EBV-positive lesions that can be found in the oral or nasal cavities, pharynx and larynx of immunosuppressed patients.Both display positivity for EBER in-situ hybridization and can be treated with conservative surgical removal.In addition, some cases might regress spontaneously with decreased immunosuppression [1,124,125].
In conclusion, a tremendous development has taken place in the classification of head and neck tumors with widespread application of molecular testing.In many cases, an expanding spectrum of immunohistochemical surrogates helps with histopathological diagnosis, and also with rapid identification of diagnostic molecular abnormalities.Therefore, pathologists currently benefit from the molecular underpinnings of tumors while still making diagnoses largely based on histology and immunohistochemistry.

Fig. 2
Fig. 2 Classical mucoepidermoid carcinoma (MEC) consisting of cystic structures with plenty of mucoid cells settled within intermediate cells (A).Clear cell variant of MEC with solid morphology and

Fig. 3
Fig.3Adenoid cystic carcinoma with cribriform to tubular morphology containing basophilic matrix and reduplicated basement membrane material (A).Solid pattern characterized by dense cellular tumour nests without pseudocystic spaces, this case showed NOTCH1

Fig. 4
Fig. 4 Hyalinizing clear cell carcinoma is characterized by clear cell morphology (A) or eosinophilic cells (B) creating solid nestes embedded within two types of stroma hylinized basement membranelike (A) and desmoplastic or fibrocellular (B).The fusions joining of

Fig. 5
Fig. 5 Microsecretory carcinoma is composed of small tubules and microcysts lined by flat intercalated duct-like cells and containing abundant basophilic luminal secretions (A).The nuclei are uniform,

Table 1
Immunohistochemical characteristics and genetic alterations and in salivary gland tumors reviewed in this article

Table 2
Sinonasal tumors reviewed in this article: Immunohistochemical features and molecular genetic data Excluded entities without diagnostic molecular genetic characteristics Adapted from: WHO Classification of Tumours Editorial Board.Head and neck tumours.Lyon (France): International Agency for Research on Cancer; forthcoming.(WHO classification of tumours series, 5th ed.; vol.9).https:// publi catio ns.iarc.fr

Table 3
Soft tissue tumors of Head and Neck reviewed in this article: Location, immunohistochemical features and molecular genetic data Excluded entities without diagnostic molecular genetic characteristics and entities not occurring predominantly in the head and neck Adapted from: WHO Classification of Tumours Editorial Board.Head and neck tumours.Lyon (France): International Agency for Research on Cancer; forthcoming.(WHO classification of tumours series, 5th ed.; vol.9).https:// publi catio ns.iarc.fr Jo VY, Demicco EG.Update from the 5th Edition of the World Health Organization Classification of Head and Neck Tumors: Soft Tissue Tumors.Head Neck Pathol.2022;16:87-100 WHO Classification of Tumours Editorial Board.Soft tissue and bone tumours.Lyon (France): International Agency for Research on Cancer; 2020.(WHO classification of tumours series, 5th ed.; vol.3).https:// publi catio ns.iarc.fr/ 588