Journal of the American Society of Cytopathology (2020) xx, 1e10

Available online at www.sciencedirect.com

ScienceDirect

journal homepage: www.jascyto.org/

HPV-related carcinoma of the oropharynx:challenges on small biopsy specimens

Doreen Palsgrove, MD, Justin A. Bishop, MD*

Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas

Received 21 April 2020; received in revised form 11 June 2020; accepted 12 June 2020

KEYWORDSHPV;Human papillomavirus;p16;Oropharynx;Squamous cell carci-noma

*Corresponding author: Justin A. Bisho633-6349; Fax: (214) 633-8817.

E-mail address: justin.bishop@utsouth

2213-2945/$36 � 2020 American Societyhttps://doi.org/10.1016/j.jasc.2020.06.001

Human papillomavirus (HPV)-related oropharyngeal carcinoma can morphologically mimic many processesfrom benign to malignant and lead pathologists to incorrectly diagnose them and/or use inappropriate diag-nostic terminology. Recognition of HPV-related oropharyngeal carcinoma and its variants is critical forappropriate prognostic and therapeutic considerations in small biopsy material. Various pitfalls in diag-nosing these tumors on limited biopsies are discussed herein.� 2020 American Society of Cytopathology.Published by Elsevier Inc. All rights reserved.

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2Oropharyngeal HPV-related SCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2HPV testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3Specific challenges in small biopsies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

HPV-related SCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5HPV-related high-grade neuroendocrine carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6Branchial cleft cyst versus metastatic oropharyngeal HPV-related SCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6Mucoepidermoid carcinoma versus HPV-related adenosquamous carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6Hyalinizing clear cell carcinoma versus oropharyngeal HPV-related SCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8Metastatic carcinoma to the oropharynx versus primary oropharyngeal HPV-related carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9Funding sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

p, MD; Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390; Tel.: (214)

western.edu (J.A. Bishop).

of Cytopathology. Published by Elsevier Inc. All rights reserved.

2 D. Palsgrove, J.A. Bishop

Conflict of interest disclosures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9Ethical approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

Introduction

Human papillomavirus (HPV) is a major etiologic factor forthe development of head and neck squamous cell carcinoma(SCC).1,2 At least 12% of pharyngeal cancer, 3% of oralcancer, and 60% to 70% of oropharyngeal carcinoma casesare caused by HPV infection, with an incidence on therise.3,4 Site for site, the prevalence of HPV-related (positive)SCC is also highest in the oropharynx, where it accounts for80% to 90% of all oropharyngeal carcinomas in Westernpopulations. The second most common site is the sinonasaltract, where 20% to 30% of SCCs have been shown to beHPV-related.5

Oropharyngeal HPV-related SCC

Transcriptionally active HPV oropharyngeal SCC is aclinically, biologically, and morphologically distinct form ofhead and neck SCC, with very few exceptions. Given theprognostic and treatment differences, it is critical for pa-thologists to accurately diagnose them and communicate thefindings according to latest iteration of the cancer stagingmanual of the American Joint Committee on Cancer.6 Assuch, it is recommended by the College of American Pa-thologists that all patients with newly diagnosed oropha-ryngeal SCC receive some form of HPV testing of theiroropharyngeal tumor tissue.7

The clinical presentation of oropharyngeal HPV-relatedSCC is relatively unique compared with other sites of headand neck HPV-related tumors. Most individuals presentwith large, cystic, painless neck masses and small, oftenclinically occult, primary lesions in the tonsil or base oftongue. The prototypical patient is a male adult aged 50 to60 years without a significant smoking history, but patientsmay be female and/or considerably younger or older.8-10

The lack of histologic evidence of a primary lesion is alsonot uncommon, such that many patients are diagnosed ashaving neck metastases of unknown primary. The proba-bility of an HPV-related metastasis of unknown primary isparticularly high in the upper and mid jugular cervicallymph nodes (synonymous with neck levels 2 and 3,respectively), which is where almost all metastatic oropha-ryngeal HPV-related SCC go.7 Of note, uncommon exam-ples of HPV-related carcinomas of the nasopharynx mayalso metastasize to cervical lymph nodes and present withclinically occult primaries. Thus, some form of high-riskHPV testing should be performed on all metastases of

unknown primary to level 2 or 3 lymph nodes so that site-directed biopsies of likely sources of disease (base oftongue, soft palate, palatine tonsils, nasopharynx) may beperformed.7 Several retrospective cohort studies have sug-gested that patients with HPV-related (high-risk HPV pos-itive) SCC in metastases of unknown primary have betteroutcomes (overall and progression-free survival) comparedwith patients with HPV unrelated (HPV-negative) SCCmetastases.7,11

Oropharyngeal HPV-related SCCs tend to have charac-teristic histologic features that are easily appreciated byroutine microscopy. This may partially be explained by theundulating epithelium native to the lymphoid tissue in thisregion that gives rise to tonsillar crypts deep within thetissue. The normal epithelium of the tonsillar crypts is asyncytial reticulated meshwork of non-stratified squamouscells permeated with tissue-resident lymphocytes (Fig. 1).The basement membrane is also notably discontinuous,which may facilitate early invasion and metastasis.12 In fact,even very small tumors that appear to be histologicallyconfined to crypt epithelium can have bulky lymph nodemetastases. As a result, all oropharyngeal HPV-relatedSCCs are regarded as invasive and capable of metastasesand differentiation is not applicable in this setting.13,14

Like the native epithelium from which it derives, proto-typical oropharyngeal HPV-related SCC is non-keratinizing,giving it a very “blue” appearance (Fig. 2). When maturingsquamous differentiation with keratinization is observed, itoften occurs on the outside of tumor cell nests in a patternthat has been referred to as “reverse maturation”.15

Oropharyngeal HPV-related SCC tends to invade assmooth-edged nests and sheets of cells with indistinct cellborders, round to oval nuclei with inconspicuous nucleoli,and a high nuclear-to-cytoplasmic ratio. The nests may alsodisplay clefting or retraction artifact. Mitotic rates aregenerally high and cystic comedo-type necrosis is frequentlyencountered, particularly in lymph node metastases. Adense lymphoplasmacytic infiltrate is typically noted in thesurrounding stroma without a significant desmoplastic re-action. Tumor fragments tend to have a syncytial qualitywith indistinct cell borders and are commonly permeated bytumor infiltrating lymphocytes. Because of the high rates ofcystic and necrotic change in lymph node metastases, it isnot uncommon for small biopsy specimens from neckmasses of unknown primaries to consist predominantly ofabundant necrotic debris and histiocytes with limited intactepithelium.

Figure 1 Similar to benign tonsillar crypt epithelium (A-C), non-keratinizing oropharyngeal HPV-related SCC (D-F) lacks significantsquamous maturation and is frequently heavily infiltrated by lymphocytes. Normal tonsillar epithelium shows patchy p16 immunostaining(C) whereas non-keratinizing oropharyngeal SCC characteristically demonstrates moderate to strong nuclear and cytoplasmic p16 staining(F). Note that the tumor-infiltrating lymphocytes are negative.

Small biopsies of HPV-related SCC 3

Many histologic variants of oropharyngeal HPV-relatedSCC have been described (eg, keratinizing, papillary,lymphoepithelial-like, adenosquamous, and sarcomatoid)and reviewed elsewhere.8,16-18 The prototypical morphology(50%-60% of cases) is non-keratinizing, in which <10% ofthe tumor shows maturing squamous differentiation. Thesetumors may still display dyskeratosis and squamous pearls.Rarely, non-keratinizing HPV-related SCC may displaystriking nuclear atypia in the form of multinucleation andfrank anaplasia.8,19 In the setting of oropharyngeal SCC,differentiation of these tumors still does not apply if they arepositive for high risk HPV. Although tempting, pathologistsshould avoid terms about grading and/or differentiation.7

For those cancers that deviate from the morphologic pro-totype of non-keratinizing squamous cell carcinoma, anassociation with HPV may not be recognized, thereby pre-cluding accurate tumor classification and staging. Forexample, for carcinomas of the head and neck that exhibitlymphoepithelial features one cannot assume an Epstein-Barr virus (EBV)-driven process by morphology alone.Luckily, most variants of oropharyngeal HPV-related SCCare not prognostically significant, with the exception of theHPV-related forms of small cell carcinoma and large cellneuroendocrine carcinoma, which are aggressive tumorswith poor outcomes.20,21 Patients with HPV-related small

cell carcinoma are also not eligible for de-escalation ther-apy. Similar to non-keratinizing oropharyngeal HPV-relatedSCC, patients with HPV-related neuroendocrine carcinomasof the head and neck frequently develop regional lymphnode metastases22 and are likely to undergo small needlecore biopsy. In many cases of HPV-related small cell car-cinoma of the oropharynx, areas of prototypical non-keratinizing HPV-SCC can be seen juxtaposed to thesmall cell areas, with both harboring integrated high-riskHPV by in situ hybridization.

HPV testing

High-risk HPV (HR-HPV) types are the causative agents ofHPV-related carcinomas in the head and neck as well as theanogenital tract. Among the HR-HPV types, HPV16 is themost common driver of oropharyngeal carcinoma and isimplicated in >90% of these patients.1,23 Because of theconsiderable prognostic significance of HPV, several pa-thology and oncology organizations recommend some formof HR-HPV testing on all oropharyngeal SCCs.7,24,25 HPVtesting is also recommended in level 2 and 3 lymph nodemetastases of unknown primary because the presence ofHPV in a neck metastasis strongly points to the ipsilateral

Figure 2 Non-keratinizing HPV-related SCC of the oropharynx (A, left side of image) often resembles the reticulated epithelium oftonsillar crypts (A, arrows) with a syncytial pattern of growth and dense lymphocytic infiltrate. Broad tumor cell nests (B), sheets (C,D), and cysts with keratin debris (E, F) may also be seen. In small biopsies, tumor fragments may be obscured by extensive cautery artifact(G). In such cases, diffuse p16 immunostaining (H) is diagnostic of malignancy.

4 D. Palsgrove, J.A. Bishop

oropharynx as the probable site of tumor origin.7 HPV-related SCCs arising outside the oropharynx (eg, oral cav-ity, larynx, hypopharynx) are uncommon and the signifi-cance of HPV in these sites is still unclear. Routine HR-HPV testing is also not recommended on patients withnon-squamous cell carcinoma of the oropharynx.7 Given thelower incidence of disease in non-oropharyngeal sites,

routine HPV testing is not recommended outside of theoropharynx at this time until clinical indications areestablished.7,26

There are many different HPV testing methods available,including p16 immunohistochemistry (IHC), DNA- orRNA-based polymerase chain reaction techniques, andDNA or RNA in situ hybridization (ISH). Note that p16

Figure 3 P16 immunohistochemistry is considered a surrogate for high-risk HPV specific testing in the oropharynx. Moderate to strongnuclear and cytoplasmic p16 immunostaining in >70% of tumor cells is considered positive (A). In situ hybridization using a high-risk HPVcocktail (HPV-16, 18, 31, 11, 53, etc.) with probes complementary to E6/E7 viral mRNA is typically used to demonstrate transcriptionallyactive high-risk HPV and allows for direct visualization in tissue. HPV-related tumors will show punctate nuclear dot-like signals in tumorcell nuclei (B, arrows), which may be focal if the viral load in the tumor is low.

Small biopsies of HPV-related SCC 5

IHC is sufficient alone as a surrogate marker of HR-HPV fororopharyngeal SCC. In this setting, nuclear and cytoplasmicp16 immunoexpression in >70% of tumor cells with at leastmoderate to strong intensity block-like staining highly cor-relates with HR-HPV detection (Fig. 3), and is thereforeregarded as positive.7 If p16 IHC is equivocal (50%-70%moderate to strong nuclear and cytoplasmic staining ordiffuse low-intensity nuclear and cytoplasmic), specificHPV testing may be considered; otherwise, the tumorshould be reported as p16-negative with a comment that thetumor is very likely negative for HR-HPV.7 If the epicenterof disease is unclear, as can be the case with multisite SCCinvolving the oropharynx, a positive p16 by IHC should befollowed up by HR-HPV testing.7 A subset of HPV-negative SCCs of the head and neck, for example, mayoverexpress p16 by other molecular mechanisms (eg,CDKN2A mutation or amplification, NSD1 mutation);therefore, p16 has a low pretest probability of HR-HPVpositivity in non-oropharyngeal SCC.7,27-29

Figure 4 This oropharyngeal carcinoma is made up of infiltrative tumratios. There is marked crush and frozen section artifact. This histologic aing for p40 reveals diffuse, strong staining (B). This finding rules out neurcarcinoma.

Specific challenges in small biopsies

HPV-related SCC

Diagnosing HPV-related SCC can be quite challenging onsmall biopsies of the oropharynx. Fortunately, biopsies ofthe oropharynx are relatively uncommon. When an HPV-related SCC is suspected, it is more typical to receivediagnostic tonsillectomies, which are easier to interpret. Partof the difficulty of diagnostic HPV-related SCC on a biopsyis based on the striking histologic similarity of HPV-relatedSCC to benign crypt epithelium native to the oropharynx.This challenge is compounded by the fact that biopsies inthis location often demonstrate crush and cautery artifact towhich non-keratinizing HPV-related SCCs are particularlysusceptible. Moreover, biopsies are often sent as frozensection specimens before a resection is performed, intro-ducing even more artifact to the biopsy.

or cords made up of cells with very high nuclear-to-cytoplasmicppearance is suspicious for small cell carcinoma (A). Immunostain-oendocrine carcinoma, and confirms the diagnosis of squamous cell

6 D. Palsgrove, J.A. Bishop

HPV-related SCC can, at times, appear virtually identicalto a normal tonsillar crypt. Helpful histologic clues to thepresence of SCC include an expansive growth pattern, ne-crosis, mitoses, and especially apoptotic bodies. Whenattempting to distinguish an HPV-related SCC from atonsillar crypt, p16 immunohistochemistry can be invalu-able. Even a very small focus of distorted HPV-SCC will bediffusely and strongly p16-positive, whereas benign cryptsare negative or patchy.

Oropharyngeal lymphoepithelial-like carcinoma is adeceptively subtle histologic variant of HPV-related SCC.Like their EBV-associated non-keratinizing undifferentiatedcounterparts in the nasopharynx, tumor cell nests and cordsare overrun and obscured by a dense nonneoplastic lym-phoplasmacytic cell infiltrate. This carcinoma can metasta-size to cervical neck lymph nodes as an occult primarycarcinoma. In such a scenario, a primary (EBV-associated)nasopharyngeal carcinoma would be the most likelyconsideration but in situ hybridiation for Epstein-Barrencoding region staining would be negative. Note that p16IHC would be diagnostic in such situations; however, theaddition of cytokeratin and/or p40 would also be helpful tofurther exclude the possibility of lymphoma.

Follicular dendritic cell sarcoma is a rare mesenchymalneoplasm that may occur in a variety of nodal sites, mostcommonly cervical lymph nodes, as well as extranodal sitessuch as the tonsil. Like non-keratinizing HPV-mediatedSCC, follicular dendritic cell sarcoma is often composed ofoval to spindled cells with indistinct cell borders, incon-spicuous nucleoli, and tumor infiltrating lymphocytes.Zhang et al reported 50% of follicular dendritic cell sar-comas showed p16 expression that met the criteria estab-lished in oropharyngeal SCC.30 Diffuse cytokeratin or p40expression is helpful to exclude follicular dendritic cellsarcoma, particularly on small biopsies.

HPV-related high-grade neuroendocrine carcinoma

A potential pitfall on small biopsies is missing an aggressivevariant of HPV-related carcinoma, namely, small cell car-cinoma or large cell neuroendocrine carcinoma. The histo-logic features of these variants are only subtly different fromclassic non-keratinizing HPV-related SCC and, as a result, apathologist must be alert to histologic clues (rosette for-mation, peripheral nuclear palisading, nuclear molding,finely dispersed salt and pepper chromatin). Whereas pro-totypical non-keratinizing HPV-related SCC is diffuselypositive for squamous markers (p40, CK5/6) and negativefor neuroendocrine markers (eg, synaptophysin), small celland large cell neuroendocrine carcinomas routinely showloss of expression or diminished p40 and CK5/6 and areconsistently positive for neuroendocrine markers. Patholo-gists should have a low threshold for screening a tumor withp40dfor example, on a tumor for which they have anysuspicion for neuroendocrine carcinoma. If the tumor isdiffusely and strongly p40 positive, neuroendocrine

carcinoma is effectively excluded (Fig. 4). If, on the otherhand, p40 is negative for only focal or weak, additionalimmunohistochemistry (eg, synaptophysin, chromogranin,INSM1, TTF1) is needed to address the possibility of anaggressive neuroendocrine carcinoma component (Fig. 5).31

Notably, when it comes to high-grade neuroendocrinecarcinomas of the head and neck, p16 immunostaining is nota suitable surrogate marker for HPV, even in theoropharynx. Like small cell and large cell neuroendocrinecarcinoma in the lung and other sites, p16 is frequentlyexpressed via mechanisms independent of HPV infection(eg, inactivation of retinoblastoma protein).20,21

Branchial cleft cyst versus metastaticoropharyngeal HPV-related SCC

In cytologic and histologic material, distinguishing meta-static oropharyngeal HPV-related SCC in a lateral necklymph node from a benign branchial cleft cyst is often quitechallenging. Although most HPV-related metastases exhibitfoci of frankly malignant cellular features, they can also bequite bland in areas. There are even rare examples of verywell-differentiated metastatic HPV-related carcinoma withgland formation and ciliated cells lining cystic spaces.32

Branchial cleft cyst epithelium, in turn, can be stratifiedsquamous or ciliated columnar with mucin production. Ifsecondarily infected, they may also demonstrate alarmingreactive cellular atypia. As a result, one must be verycautious about dismissing a seemingly bland cystic lesion asa branchial cleft cyst, particularly in a patient over 40 yearsold.

Ultimately, demonstrating integrated HPV is crucial inmaking this diagnostic distinction. In this setting the pres-ence of high-risk HPV is diagnostic of malignancy (Fig. 6).One caveat to this is the use of p16 immunohistochemistry.Branchial cleft cysts are often rather diffusely positive forp16.33 Therefore, p16 is not a reliable surrogate marker forHPV infection if a branchial cleft cyst is part of the differ-ential diagnosis. In these cases, a more specific HPV testingmethod (eg, ISH) must be used to confirm malignancy.

Mucoepidermoid carcinoma versus HPV-relatedadenosquamous carcinoma

Minor salivary gland tumors occasionally arise in theoropharynx and metastasize to cervical lymph nodes, themost common being mucoepidermoid carcinoma (MEC).34

MEC is classically composed of 3 distinct cell types(mucinous, epidermoid, and intermediate cells) and char-acterized by MAML2 translocation in the majority of cases,including ciliated and Warthin-like variants.35 It is oftenunder-recognized that in HPV-related adenosquamous car-cinoma, mucoepidermoid morphology may be encountered.These carcinomas arise not only from the oropharynx, butalso the sinonasal tract. In contrast to conventional, HPV-

Figure 5 HPV-related small cell carcinoma (A, B) demonstrates histologic features characteristic of small cell carcinoma encountered inother organ sites (small monotonous cells with minimal cytoplasm, hyperchromatic nuclei without nucleoli, nuclear molding, abundantapoptotic debris, brisk mitotic rate). A subset of cases may also retain a component of classic non-keratinizing HPV-related SCC (A, rightside of image), which, in contrast to the small cell carcinoma, would demonstrate finely dispersed chromatin and indistinct cell borders.Classic non-keratinizing HPV-related SCC would also be diffusely positive for p40 (B, right side of image) while the small cell carcinomacomponent would be negative (B, left side of image) or only focally positive. HPV-related large cell neuroendocrine carcinomas (C, D) oftengrow as nests and trabeculae with peripheral palisading of basophilic tumor cells. The tumor cells typically contain varying amounts of cyto-plasm, coarse chromatin, and prominent nucleoli. Sometimes, an intermingled HPV-related squamous cell component can be identified (E,F). In this example, keratin pearl formation is easily identified (E), but the tumor shows only patchy p40 staining (F). Helpful immunostainsfor any high grade neuroendocrine carcinoma include: synaptophysin (G1), chromogranin (G2), TTF-1 for small cell carcinoma (G3), andCD56 (G4).

Small biopsies of HPV-related SCC 7

unrelated adenosquamous carcinoma, prominence of non-keratinizing squamoproliferative areas resembling epider-moid and even intermediate cells, admixed mucocytes andcystic spaces rather than separate, discrete tubuloglandularstructures, and scattered columnar or even ciliated cells mayall be seen in HPV-related adenosquamous carcinoma of theoropharynx (Fig. 6).32,36 Pronounced keratinization, multi-nucleation, and nuclear atypia, which are useful histologicclues in separating HPV-related adenosquamous carcinoma

from MEC (which is relatively monotonous even in high-grade examples), may also be absent, especially in biopsymaterial. The distinction is not trivial, as MEC would nottypically be treated with chemoradiation alone, as HPV-related carcinomas often are.

Ultimately, separating MEC from HPV-related adenos-quamous carcinoma requires HPV testing, which must bedone on any high-grade “mucoepidermoid-like” tumorarising in the oropharynx or sinonasal tract. Although p16

Figure 6 Example of a neck cyst lined by proliferative squamous and pseudostratified columnar epithelium with apical cilia (A, B). Thetumor was positive for high-risk HPV by RNA in situ hybridization in both the squamous and ciliated glandular components (C). This HPV-related adenosquamous carcinoma resembled mucoepidermoid carcinoma, with an eosinophilic appearance, microcysts, monotonous cellsresembling intermediate cells, and scattered mucocytes (D, E) which were positive for mucicarmine (E, inset). The tumor was positivefor high risk HPV by RNA in situ hybridization in all cell types (F).

8 D. Palsgrove, J.A. Bishop

IHC may be positive in mucoepidermoid carcinoma, espe-cially if high grade,37 specific HPV testing by ISH will benegative in tumor cell nuclei even in the absence of MAML2translocation.38 While MAML2 analysis is helpful if posi-tive (confirming a MEC diagnosis), it is important toremember that not all MECs harbor this alteration, so anegative result is not helpful in this differential diagnosis.

Hyalinizing clear cell carcinoma versusoropharyngeal HPV-related SCC

Another minor salivary gland tumor with morphologicoverlap is hyalinizing clear cell carcinoma (HCCC). HCCCis a low grade neoplasm that commonly arises in the base oftongue or palate and is characterized by EWSR1-ATF1 genefusion.39 Contrary to its name, HCCC may display aprominent eosinophilic rather than clear cell appearance,have areas of overt keratinization, and lack stromal hyali-nization. Further complicating the distinction of HCCCfrom HPV-related SCC is the presence of desmoplasia in asubset of HPV-related SCC. The squamous markers CK5/6and p40 are also diffusely positive in HCCC, furthercomplicating this dilemma.

A well-meaning pathologist encountering a p40-positivesquamoid-appearing tumor in the oropharynx (where HCCCcommonly occurs) may mistakenly order p16 as the Collegeof American Pathologists guidelines indicate should be donefor an oropharyngeal SCC. Unfortunately, HCCC has beenshown to demonstrate p16 immunoexpression with a subsetmeeting the criteria of positivity for oropharyngeal HPV-related SCC.40 This pitfall has significant clinical conse-quences, as HCCC is a low-grade tumor that must be treated

with surgery and does not respond to the chemotherapy andradiation typically utilized for HPV-related SCC.

Fortunately, simply being aware that HCCC is a squa-moid tumor that often arises in the oropharynx is usuallysufficient to avoid this misstep. As a low-grade tumor,HCCC lacks necrosis and basaloid features, and demon-strates nuclear monotony and a low mitotic rate, featuresthat are very different than most HPV-related SCCs andallow separation on routine histology alone. If HCCC issuspected, EWSR1 fluorescence in situ hybridization canconfirm the diagnosis, as it is positive in most cases.

Metastatic carcinoma to the oropharynx versusprimary oropharyngeal HPV-related carcinoma

Pathologists should consider the possibility of metastaticcancer when examining an oral/oropharyngeal mass. Rarely,oral metastases may be the first sign of metastatic spreadand/or the first indication of an undiscovered malignancy ata distant site. The most common metastatic site in the oralcavity is the gingiva and alveolar mucosa, followed by thetongue, and with much less frequency the tonsil, palate, lip,buccal mucosa and floor of the mouth.41 The most commonprimary cancers to metastasize to the oral region includelung, genitourinary tract, liver, prostate, and breast.41 Withheightened awareness, appropriate diagnostic methods,including expanded immunohistochemical panels, should becarried out in these cases. It should be remembered that rareHPV-related oropharyngeal carcinomas can have a promi-nent (ie, adenosquamous) or even exclusive (ie, adenocar-cinoma) glandular component.42 As a result, for anoropharyngeal carcinoma with an unusual glandular

Small biopsies of HPV-related SCC 9

appearance for which distant metastasis is considered,finding the tumor to harbor high-risk HPV is helpful ineffectively confirming that it is primary to that site.

Summary

p16 immunostaining should not be performed in isolationand used as unequivocal evidence of an HPV-related SCC.For carcinomas showing prominent squamous differentia-tion by morphology, or unusual morphology that divergesfrom the prototypical non-keratinizing oropharyngeal HPV-related SCC, specific HPV testing needs to be performedbefore considering a patient as having HPV-related carci-noma. Even within the oropharynx, pathologists should beaware of rare mimickers of HPV-related SCC that are alsop16-positive. Therefore, a panel of immunostains may benecessary, particularly in small biopsy material and/or forpathologists who do not see these other entities with regu-larity. These mimickers serve as a reminder that p16 posi-tivity should not be used alone as a diagnostic marker.

Funding sources

None.

Conflict of interest disclosures

All authors declare that he/she has no conflict of interest asit relates to this research project.

Ethical approval

Not applicable.

References

1. Gillison ML, Koch WM, Capone RB, et al. Evidence for a causal as-sociation between human papillomavirus and a subset of head and neckcancers. J Natl Cancer Inst. 2000;92:709e720.

2. Ang KK, Harris J, Wheeler R, et al. Human papillomavirus and sur-vival of patients with oropharyngeal cancer. N Engl J Med. 2010;363:24e35.

3. Senkomago V, Henley SJ, Thomas CC, et al. Human papillomavirus-attributable cancersdUnited States, 2012-2016. MMWR Morb MortalWkly Rep. 2019;68:724e728.

4. Van Dyne EA, Henley SJ, Saraiya M, et al. Trends in humanpapillomavirus-associated cancerdUnited States, 1999-2015. MMWRMorb Mortal Wkly Rep. 2018;67:918e924.

5. Oliver JR, Lieberman SM, Tam MM, et al. Human papillomavirus andsurvival of patients with sinonasal squamous cell carcinoma. Cancer.2020;126:1413e1423.

6. Amin MB. American Joint Committee on Cancer, American CancerSociety. AJCC Cancer Staging Manual. Chicago, IL: American JointCommittee on Cancer, Springer; 2017.

7. Lewis Jr JS, Beadle B, Bishop JA, et al. Human papillomavirus testingin head and neck carcinomas: guideline from the College of AmericanPathologists. Arch Pathol Lab Med. 2018;142:559e597.

8. Thompson LDR, Burchette R, Iganej S, et al. Oropharyngeal squamouscell carcinoma in 390 patients: analysis of clinical and histologicalcriteria which significantly impact outcome. Head Neck Pathol;2019.

9. Sinno S, Assaad AM, Salem Shabb N. Human papillomavirus-associated oropharyngeal high-grade neuroendocrine carcinoma in anadolescent: case report and review of literature. Clin Med InsightsPediatr. 2019;13. 1179556519870520.

10. Fakhry C, Waterboer T, Westra WH, et al. Distinct biomarker andbehavioral profiles of human papillomavirus-related oropharynx cancerpatients by age. Oral Oncol. 2020;101:104522.

11. Schroeder L, Boscolo-Rizzo P, Dal Cin E, et al. Human papillomavirusas prognostic marker with rising prevalence in neck squamous cell car-cinoma of unknown primary: a retrospective multicentre study. Eur JCancer. 2017;74:73e81.

12. Perry ME. The specialised structure of crypt epithelium in the humanpalatine tonsil and its functional significance. J Anat. 1994;185:111e127.

13. Randall DA, Johnstone PA, Foss RD, et al. Tonsillectomy in diagnosisof the unknown primary tumor of the head and neck. Otolaryngol HeadNeck Surg. 2000;122:52e55.

14. Pai SI, Westra WH. Molecular pathology of head and neck cancer: im-plications for diagnosis, prognosis, and treatment. Annu Rev Pathol.2009;4:49e70.

15. Lewis Jr JS, Khan RA, Masand RP, et al. Recognition of nonkeratiniz-ing morphology in oropharyngeal squamous cell carcinomada pro-spective cohort and interobserver variability study. Histopathology.2012;60:427e436.

16. Devins KM, Tetzlaff MT, Baloch Z, et al. The evolving landscape ofHPV-related neoplasia in the head and neck. Hum Pathol. 2019;94:29e39.

17. Lewis Jr JS. Morphologic diversity in human papillomavirus-relatedoropharyngeal squamous cell carcinoma: catch me if you can!. ModPathol. 2017;30:S44eS53.

18. Holmes BJ, Wenig BM. Virus-associated carcinomas of the head &neck: update from the 2017 WHO classification. Ann Diagn Pathol.2019;38:29e42.

19. Lewis Jr JS, Scantlebury JB, Luo J, et al. Tumor cell anaplasia andmultinucleation are predictors of disease recurrence in oropharyngealsquamous cell carcinoma, including among just the humanpapillomavirus-related cancers. Am J Surg Pathol. 2012;36:1036e1046.

20. Bishop JA, Westra WH. Human papillomavirus-related small cell car-cinoma of the oropharynx. Am J Surg Pathol. 2011;35:1679e1684.

21. Thompson ED, Stelow EB, Mills SE, et al. Large cell neuroendocrinecarcinoma of the head and neck: a clinicopathologic series of 10cases with an emphasis on HPV status. Am J Surg Pathol. 2016;40:471e478.

22. Jo VY, Krane JF, Pantanowitz L, et al. HPV-associated neuroendocrinecarcinomas of the head and neck in FNA biopsies: clinicopathologicfeatures of a rare entity. Cancer Cytopathol. 2019;127:26e34.

23. Walline HM, Komarck C, McHugh JB, et al. High-risk human papillo-mavirus detection in oropharyngeal, nasopharyngeal, and oral cavitycancers: comparison of multiple methods. JAMA Otolaryngol HeadNeck Surg. 2013;139:1320e1327.

24. Adelstein D, Gillison ML, Pfister DG, et al. NCCN Guidelines insights:head and neck cancers, version 2.2017. J Natl Compr Canc Netw.2017;15:761e770.

25. Fakhry C, Lacchetti C, Rooper LM, et al. Human papillomavirustesting in head and neck carcinomas: ASCO clinical practice guidelineendorsement of the College of American Pathologists guideline. J ClinOncol. 2018;36:3152e3161.

26. Fakhry C, Westra WH, Wang SJ, et al. The prognostic role of sex, race,and human papillomavirus in oropharyngeal and nonoropharyngealhead and neck squamous cell cancer. Cancer. 2017;123:1566e1575.

27. Sundberg J, Korytowska M, Burgos PM, et al. Combined testing ofp16 tumour-suppressor protein and human papillomavirus in patients

10 D. Palsgrove, J.A. Bishop

with oral leukoplakia and oral squamous cell carcinoma. AnticancerRes. 2019;39:1293e1300.

28. Lim AM, Do H, Young RJ, et al. Differential mechanisms of CDKN2A(p16) alteration in oral tongue squamous cell carcinomas and correla-tion with patient outcome. Int J Cancer. 2014;135:887e895.

29. Lechner M, Chakravarthy AR, Walter V, et al. Frequent HPV-independent p16/INK4A overexpression in head and neck cancer.Oral Oncol. 2018;83:32e37.

30. Zhang L, Yang C, Lewis Jr JS, et al. p16 expression in follicular den-dritic cell sarcoma: a potential mimicker of human papillomavirus-related oropharyngeal squamous cell carcinoma. Hum Pathol. 2017;66:40e47.

31. Lewis Jr JS, Chernock RD, Bishop JA. Squamous and neuroendocrinespecific immunohistochemical markers in head and neck squamous cellcarcinoma: a tissue microarray study. Head Neck Pathol. 2018;12:62e70.

32. Bishop JA, Westra WH. Ciliated HPV-related carcinoma: a well-differentiated form of head and neck carcinoma that can be mistakenfor a benign cyst. Am J Surg Pathol. 2015;39:1591e1595.

33. Cao D, Begum S, Ali SZ, et al. Expression of p16 in benign and malignantcystic squamous lesions of the neck. Hum Pathol. 2010;41:535e539.

34. Goel AN, Badran KW, Braun APG, et al. Minor salivary gland carci-noma of the oropharynx: a population-based analysis of 1426 patients.Otolaryngol Head Neck Surg. 2018;158:287e294.

35. Bishop JA, Cowan ML, Shum CH, et al. MAML2 rearrangements invariant forms of mucoepidermoid carcinoma: ancillary diagnostic

testing for the ciliated and warthin-like variants. Am J Surg Pathol.2018;42:130e136.

36. Radkay-Gonzalez L, Faquin W, McHugh JB, et al. Ciliated adenosqu-amous carcinoma: expanding the phenotypic diversity of humanpapillomavirus-associated tumors. Head Neck Pathol. 2016;10:167e175.

37. Jour G, West K, Ghali V, et al. Differential expression of p16(INK4A)and cyclin D1 in benign and malignant salivary gland tumors: a studyof 44 Cases. Head Neck Pathol. 2013;7:224e231.

38. Bishop JA, Yonescu R, Batista D, et al. Mucoepidermoid carcinomadoes not harbor transcriptionally active high risk human papillomaviruseven in the absence of the MAML2 translocation. Head Neck Pathol.2014;8:298e302.

39. Antonescu CR, Katabi N, Zhang L, et al. EWSR1-ATF1 fusion is anovel and consistent finding in hyalinizing clear-cell carcinoma of sali-vary gland. Genes Chromosomes Cancer. 2011;50:559e570.

40. Bishop JA, Rooper LM, Chiosea SI, et al. Clear cell carcinoma of sali-vary glands is frequently p16 positive: a pitfall in the interpretation oforopharyngeal biopsies. Am J Surg Pathol. 2018;42:367e371.

41. Hirshberg A, Shnaiderman-Shapiro A, Kaplan I, et al. Metastatic tu-mours to the oral cavitydpathogenesis and analysis of 673 cases.Oral Oncol. 2008;44:743e752.

42. Chang AM, Nikiforova MN, Johnson JT, et al. Human papillomavirus-associated adenocarcinoma of the base of tongue: potentially actionablegenetic changes. Head Neck Pathol. 2014;8:151e156.