related small cell carcinoma of the oropharynx

Original Article

Cytopathologic Characteristics of HPV-Related Small Cell Carcinoma of the Oropharynx Derek B. Allison, MD1; Lisa M. Rooper, MD2; Sara Mustafa, MD1; Zahra Maleki, MD1 Paul E. Wakely Jr, MD3; and Syed Z. Ali, MD4

;

BACKGROUND: Human papillomavirus (HPV)–related squamous cell carcinoma (SqCC) of the oropharynx is an epidemiologically and clinically distinct form of SqCC that is associated with an improved prognosis. However, HPV-related small cell carcinoma of the oropharynx is a rare and newly described variant that is associated with aggressive clinical behavior and poor outcomes. To date, fewer than 2 dozen reports of this entity exist in the literature, and there is no discussion of cytopathologic features. This article reports 6 cases and discusses the salient cytomorphologic findings, ancillary studies, and challenges when this entity is encountered. METHODS: Anatomic pathology archives were searched to identify patients with a diagnosis of HPV-related small cell carcinoma of the oropharynx. Medical records were reviewed to document the following: age, sex, smoking status, other relevant clinical history, primary location, treatment, and clinical outcome. Both p16 and high-risk HPV in situ hybridization (ISH) studies were positive in at least 1 specimen from each patient. The pathologic diagnoses, cytomorphologic characteristics, immunocytochemical stains, and HPV ISH studies were reviewed and recorded for all available cases. RESULTS: Six patients with 11 cytopathology specimens of HPV-related small cell carcinoma of the oropharynx were identified. The mean age was 61.3 years, and all patients died with widely metastatic disease (mean, 23 months; range, 12-48 months). Mixed small cell carcinoma and SqCC components were present in half of the cases. CONCLUSIONS: The identification of a small cell component can be reliably performed with cytology preparations and is crucial because this (and not the HPV status) determines the prognosis. Cancer Cytopathol 2018;0:1-9. © 2018 American Cancer Society. KEY WORDS: cytopathology; head and neck cancer; human papillomavirus; small cell carcinoma; squamous cell carcinoma.

INTRODUCTION Human papillomavirus (HPV)–related squamous cell carcinoma (SqCC) of the oropharynx is an epidemiologically and clinically distinct subtype of head and neck SqCC that has increased in incidence in recent decades.1,2 Patients with HPV-related SqCC are generally younger than patients with conventional SqCC, are more frequently nonsmokers, and often present with small palatine tonsil or base of tongue primaries and early neck lymph node metastases.1,3 Importantly, HPV-related SqCC is associated with better overall survival with an improved response to chemotherapy and radiation treatment in comparison with conventional SqCC.4–6 As a result, HPV testing has become routine practice for oropharyngeal SqCC because it has significant implications for prognosis and clinical trial enrollment.7,8 Several histologic variants of HPV-related oropharyngeal carcinoma, including basaloid, papillary, lymphoepithelial-like, adenosquamous, and sarcomatoid SqCC variants, have been identified.9,10 Despite the variation in the morphologic appearance or high-risk (HR) features associated with these tumor types in conventional

Corresponding author: Derek B. Allison, MD, Department of Pathology, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21287; [email protected] 1

Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland; 2Department of Pathology and Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland; 3Department of Pathology, Ohio State University Wexner Medical Center, Columbus, Ohio; 4 Department of Pathology and Radiology, Johns Hopkins University School of Medicine, Baltimore, Maryland Received: August 12, 2018; Revised: September 19, 2018; Accepted: October 4, 2018 Published online Month 0, 2018 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cncy.22078 , wileyonlinelibrary.com

Cancer Cytopathology Month 2018

1

Original Article

SqCC, these variants seem to impart the same favorable prognosis for HPV-related tumors.9–12 In 2011, however, Bishop and Westra13 described HPV-related oropharyngeal small cell carcinomas. In contrast to SqCC variants, HPV-related small cell carcinomas follow an aggressive clinical course and are almost uniformly fatal. To date, fewer than 2 dozen cases of HPV-related small cell carcinoma of the oropharynx have been reported in the literature.13–20 Despite the growing use of fine-needle aspiration (FNA) cytology for the evaluation of neck masses and masses at other sites and the adequate performance of these specimens for HPV testing, there are no reports in the literature describing the cytomorphologic features of this entity.21–23 Here we report the first study of HPVrelated small cell carcinoma of the oropharynx in cytology preparations.

MATERIALS AND METHODS The Johns Hopkins institutional review board approved this study. We searched the anatomic pathology archives of the Johns Hopkins Hospital and the Ohio State University Wexner Medical Center to identify patients with a diagnosis of HPV-related small cell carcinoma of the oropharynx. All available cytopathology specimens containing diagnostic material of this entity were reviewed and included in this study, and all patients had p16 and HPV in situ hybridization (ISH) positivity on at least 1 cytopathology or surgical pathology specimen. The medical records were reviewed to document the following: age, sex, smoking status, other relevant clinical history, primary location, treatment, and outcome. The pathologic diagnoses, cytomorphologic characteristics, immunocytochemical stains, and ISH studies were reviewed and recorded for all available cases. The overall specimen cellularity was assessed with a 4-point scale like the one reported by Othman et al24 and used previously by Allison et al25 as follows: 1 = scant, 2 = hypocellular, 3 = moderately cellular, and 4 = hypercellular. Cytopathology Material

At the time of the original diagnosis, all FNA was performed under ultrasound guidance with a rapid onsite evaluation performed by a cytotechnologist or cytopathologist. Aspirations were performed with 25-gauge spinal needles, and small core biopsies were performed with a 20-gauge biopsy device. Multiple FNA passes were performed, and when it was feasible, core biopsy 2

specimens were obtained. Aspirated material was immediately smeared onto glass slides and either air-dried or alcohol-fixed with 95% ethanol. In a subset of cases, material remaining from the needle was rinsed with Hanks balanced salt solution to make a formalin-fixed, paraffin-embedded cell block. Air-dried smears and touch preparations were stained with the Mercedes Medical Platinum Line Quik-Dip fixative, alcohol-fixed smears were stained with the Papanicolaou method, and cell blocks and small core biopsy specimens were stained with hematoxylin-eosin. Immunohistochemistry (IHC)

IHC stains were performed on 5-µm sections prepared from formalin-fixed, paraffin-embedded cell blocks or core biopsy material. Standard autostaining protocols on a Ventana Benchmark XT autostainer (Ventana Medical Systems, Inc, Tucson, Arizona) were followed. Positive and negative controls were appropriate and were performed with each run. A mouse monoclonal antibody for p16 (MTM Laboratories, Heidelberg, Germany) was used. For core biopsies, p16 expression was considered positive if ≥70% of the tumor cells showed strong, diffuse nuclear and cytoplasmic staining26; >50% staining was considered to be positive for p16 expression in cell block material on the basis of the authors’ anecdotal experience, although some studies have suggested that as little as 15% staining in cell block material may be considered positive for p16 expression.23,27,28 In Situ Hybridization

In situ hybridization studies were performed on 5-µm sections prepared from formalin-fixed, paraffin-embedded core biopsy material or excisional material, as previously described.29,30 For cases diagnosed after November 2014, the presence of HR HPV was assessed by the detection of HR HPV E6/E7 messenger RNA with the RNAscope HPV-HR18 Probe (Advanced Cell Diagnostics), which recognizes 18 HR HPV genotypes (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82). Studies with punctate cytoplasmic and/ or nuclear signals were considered positive. For cases diagnosed before November 2014 or diagnosed at the Ohio State University Wexner Medical Center, the presence of HR HPV was assessed with the DNA ISH HPV III Family 16 Probe (B) kit (Ventana Medical Systems), which recognizes 12 HR HPV genotypes (16, 18, 31, Cancer Cytopathology Month 2018


48 DWD S/CT/RT HPV DNA ISH–positive Positive SCC Tonsil NA 35 pack-y 48 6

Male

64 5

Abbreviations: Tx, treated; ADT, androgen-deprivation therapy; BOT, base of tongue; CC-RCC, clear cell renal cell carcinoma; CT, chemotherapy; DWD, died with disease; HPV, human papillomavirus; ISH, in situ hybridization; LCNC, large cell neuroendocrine carcinoma; NA, not applicable; RT, radiation therapy; S, surgery; SCC, small cell carcinoma; SmCC, small cell carcinoma; SqCC, squamous cell carcinoma.

14 DWD CT/RT/ nivolumab HPV RNA ISH–positive Positive BOT mass never sampled BOT mass Tonsillectomy as child Never

69 4

Male

26 DWD CT/RT/ nivolumab HPV RNA ISH–positive Positive HPV-positive combined SmCC and SqCC Tonsil Prostatic adenocarcinoma, grade group 5 on ADT; Tx CC-RCC 30 pack-y

56 3

Male

16 DWD S/CT/RT HPV RNA ISH–positive Positive HPV-positive combined SmCC and SqCC Tonsil NA Never

56 2

Male

12 DWD CT/RT HPV RNA ISH–positive Positive HPV-positive combined SmCC, LCNC, and SqCC BOT NA Never

81 1

Male

Not available Not available HPV RNA ISH–positive Positive HPV-positive SmCC BOT Tx prostatic adenocarcinoma, grade group 1

HPV-Specific Confirmation p16 Primary Diagnosis Primary Location Other Diagnosis Smoking Status Sex Age, y

Of the 11 cytopathology specimens identified, 10 were available for review. The cytomorphologic characteristics are summarized in Table 2 and displayed in Figure 1.

Patient

Cytopathologic Findings

TABLE 1. Patient Characteristics

Six patients with 11 cytopathology specimens of HPVrelated small cell carcinoma of the oropharynx were identified. One patient’s clinical presentation was previously described in a case report.31 Ten specimens were from FNA procedures, whereas 1 specimen was from a tonsillar imprint performed at the time of surgery. The patient characteristics are summarized in Table 1. Briefly, patients ranged in age from 48 to 81 years (mean, 61.3 years) at the time of their first diagnosis. Four patients (66.7%) were never smokers, whereas 2 had smoking histories (30 and 35 pack-years). Cytology specimens were the first diagnostic specimens obtained for 4 patients (66.7%). Three primary lesions (50.0%) occurred in the tonsil, whereas 3 (50.0%) occurred in the base of tongue. Five patients (83.3%) also underwent biopsy or resection of their primary tumor, a histologic review of which showed 3 combined tumors with small cell carcinoma (with 1 also containing large cell neuroendocrine features) and SqCC components (60.0%) and 2 tumors that contained only small cell carcinoma (40.0%). The patient whose primary tumor did not undergo histologic sampling had metastases at different time courses with both combined features and only small cell features. The reviewed FNA specimens included aspirates from 6 cervical lymph nodes, 3 liver metastases, and 1 back mass metastasis. Treatment and follow-up data were available for 5 patients (83.3%). All patients were treated with chemotherapy and radiation therapy; 2 (40.0%) additionally received surgical debulking excisions, whereas 2 also were treated with nivolumab, an anti–programmed death 1 monoclonal antibody. All patients died with widely metastatic disease 12 to 48 months after their diagnosis (mean, 23 months).

Treatment

Clinical Characteristics

Never

RESULTS

Male

Outcome

Interval, mo

33, 35, 39, 45, 51, 52, 56, 58, and 66). Studies with punctate signals present in the nucleus were considered positive.

NA

HPV-Related Small Cell Carcinoma/Allison et al

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Original Article

Briefly, the specimens were highly cellular (mean cellularity, 3.3/4) and were composed of sheets of single cells and clusters in a background of necrotic debris and numerous apoptotic bodies. Mitoses were frequent, and classic features of small cell carcinoma, including a nuclear crush artifact, nuclear molding, and finely granular, salt and pepper chromatin, were present in all cases with appropriate cellularity. One case with scant cellularity (1 of 4) did not contain a mitosis, most likely because of a limited amount of intact, nonnecrotic cellular material. Half of the cases contained a separate cell population that had squamous differentiation, mostly in syncytial fragments and often with a basaloid phenotype, or consisted of larger, oval-to-spindled epithelial cells with more but overall scant hard cytoplasm and intermediatesized, hyperchromatic nuclei with inconspicuous nucleoli; keratinization was absent in all cases. In all cases with combined small cell and squamous cell features, the small cell component was the predominant population; however, the squamous cell population was readily identified. Tumor heterogeneity was apparent because of the varying proportions of these 2 cell populations from pass to pass. IHC Studies

IHC stains were performed on 4 core biopsy specimens and 1 cell block. For the case with a tonsillar imprint, these studies were performed on the tonsillar excision material. The IHC results for each of these cases is presented in Table 2, and representative IHC stains are shown in Figure 2. Briefly, pankeratin cocktails and p16 were positive in all tested cases. Synaptophysin was positive in 4 of 5 cases (80.0%), chromogranin was positive in 2 of 5 cases (40.0%), and CD56 was positive in 3 of 4 cases (75.0%). p40 was performed in 5 cases: it was positive in the 2 cases with morphologic evidence of squamous differentiation on the smears and negative in the 3 cases without evidence of squamous differentiation on the smears. Thyroid transcription factor 1 was performed, and the results were negative in 2 of 2 specimens. Ki-67 was performed in 3 specimens, all of which demonstrated a proliferative index higher than 90%. In Situ Hybridization Findings

All patients included in this study had positive HR HPV– specific testing performed on at least 1 of their FNA or excisional specimens. Four patients had HR HPV RNA 4

ISH studies only, 1 patient had an HR HPV DNA ISH study only, and 1 patient had both performed on different specimens. There were 11 FNA specimens presented in this study, and HR HPV–specific testing was performed for 5 cases (45.5%); 4 were core biopsy specimens, and 1 was a tonsillar excision (the tonsillar imprint case). HR HPV RNA ISH was positive in all 5 tested cases. In 1 case, HR HPV DNA ISH was equivocal; however, HR HPV RNA ISH was positive on the corresponding excision material. HR HPV DNA ISH was positive in the other tested case.

DISCUSSION HPV-related small cell carcinoma of the oropharynx is a newly recognized variant with few cases reported in the literature.13–19 This study presents the first comprehensive cytopathologic characterization of this entity and includes 6 patients. Overall, the presentation and clinical course of these tumors were similar to those in previous reports. As in other series, tobacco was not a consistent risk factor for developing HPV-related small cell carcinoma, with two-thirds of the patients never being smokers. Likewise, similarly to tumors in prior reports, all tumors in this study demonstrated aggressive behavior, with all patients dying of disease within 12 to 48 months of the time of their first diagnosis. Also, as in previous reports, 3 primary tumors (60.0%) that were sampled histologically demonstrated features of combined small cell carcinoma and SqCC. In our cytology samples, half of the cases showed evidence of both small cell and squamous differentiation; however, interestingly, all 4 samples from distant metastatic sites contained only small cell carcinoma components, even when primary and regional metastases from these patients contained combined features. Conversely, all cases of combined small cell and squamous differentiation were metastases located in the neck. The presence of small cell carcinoma exclusively in distant metastases suggests that this component drives the aggressive clinical course. FNA smears seem to reliably detect small cell and squamous cell differentiation without the need for IHC. This finding is important because these 2 entities do have some overlapping features. Because HPV-related SqCC is typically nonkeratinizing or minimally keratinizing and has an immature, basaloid phenotype with many mitoses and areas of necrosis, it can potentially Cancer Cytopathology Month 2018

Case

1

2

3

4

5

6

7

8

9

10

Patient

1

2

3

3


4

4

5

5

5

5

Back mass

Neck 2A/3 LN

Neck mass

Neck mass

Liver

Neck 2A LN

Liver

Neck 2B LN

Neck LN

Liver

Site

Poorly differentiated carcinoma with small cell features

Predominantly necrotic cells and debris

Metastatic SmCC

Malignant neoplasm with extensive necrosis; differential includes SmCC and SqCC

Metastatic SmCC

Metastatic HG basaloid carcinoma with myoepithelial and focal NE differentiation

Metastatic SmCC

Metastatic SmCC

Metastatic NSmCC

Metastatic HPV-positive SmCC

Diagnosis

Cytopathology

NAR

Scant single cells and rare clusters Sheets of single cells and clusters

1

4

Predominantly clusters

3

NAR

Sheets of single cells and clusters


3

4

Sheets of single cells

Predominantly clusters

Scant single cells and rare clusters


Architecture

4

4

2

4

Cellularitya

TABLE 2. Cytomorphologic Characteristics

Present

Present (abundant)

NAR

Present (abundant)

Present (scant)

Present

Present

Present

Present (scant)

Present (scant)

Background

Necrotic

Present

Absent

Present

Present

NAR

Present

Present

NAR

Present

Present

Present (abundant)

NAR

Present

Present

Present

Present

Present

Present

Differentiation

Small Cell/NE

Present

Present

Present

Present

Present

Present

Mitosis

Present (scant)

Present

Present

Present

Present (scant)

Present

Apoptosis

Absent

Present

NAR

Present

Absent

Present

Absent

Absent

Present (focal)

Absent

Differentiation

Squamous

Absent

Absent

NAR

Absent

Absent

Absent

Absent

Absent

Absent

Absent

Keratinization

HPV: HR HPV RNA ISH–positive (on prior FNA specimen)

HPV: HR HPV RNA ISH–positive (on prior FNA specimen)

Positive: p16, synaptophysin Negative: chromogranin, p40, napsin A HPV: HR HPV RNA ISH–positive

HPV: HR HPV RNA ISH–positive (performed on excisional material)


Positive: CD56, synaptophysin (focal), chromogranin (focal), p40 Negative: NKX3.1, PSA, P501S, TTF1, PAX-8 HPV: HR HPV RNA ISH–positive (performed on excisional material)


Positive: AE1/AE3, Cam 5.2, p16, synaptophysin, CD56, Ki-67 (>95%) Negative: p40, CK20, chromogranin HPV: HR HPV DNA ISH–equivocal; HR HPV RNA ISH–positive (performed on excisional material)

Positive: AE1/AE3, p63 (focal), p40 (focal), p16 HPV: HR HPV RNA ISH–positive (performed on excisional material)

Positive: p16, Ki-67 > 90% Negative: p40, chromogranin, synaptophysin, CD56 HPV: HR HPV RNA ISH–positive

Testing

IHC/HPV


5

6

Abbreviations: EBER, Epstein-Barr encoding region; FNA, fine-needle aspiration; HG, high-grade; HPV, human papillomavirus; HR, high-risk; IHC, immunohistochemistry; ISH, in situ hybridization; LN, lymph node; NAR, not available for review; NE, neuroendocrine; NSmCC, non–small cell carcinoma; PAX-8, paired box 8; PSA, prostate-specific antigen; SmCC, small cell carcinoma; TTF1, thyroid transcription factor 1. a Cellularity was classified on a scale from 1 to 4, with 1 indicating paucicellularity and 4 indicating hypercellularity. b Immunohistochemical stains and HR HPV DNA ISH were performed on excisional material.

Testing

Positive: AE1/AE3, CK-OSCAR, p16, p63, CD56, synaptophysin, chromogranin, bcl-2, Ki-67 (>99%)b Negative: CD3, CD5, CD20, bcl-6, myoD1, CDX-2, TTF1, EBER ISH HPV: HR HPV DNA ISH–positive Absent Present Present Present Present Present Sheets of single cells and clusters 4 Small cell NE carcinoma 11

Tonsil, imprint

Differentiation Differentiation Background

6

Cytopathology

Diagnosis Site Case Patient

TABLE 2. (Continued)

Cellularitya

Architecture

Necrotic

Apoptosis

Mitosis

Small Cell/NE

Squamous

Keratinization

IHC/HPV

Original Article

mimic small cell carcinoma. True small cell carcinomas, however, usually have more crush artifact, nuclear molding, and characteristic neuroendocrine-type chromatin. In our study, all IHC results were consistent with the lineages observed on the smears. Not only does this finding confirm the fidelity of the cytomorphologic impressions of these cases, but it also suggests that IHC may be helpful for confirming the cytomorphologic findings if necessary. Although p40 is an excellent marker for confirming squamous differentiation, p63 has been shown to be positive in some small cell carcinomas of the head and neck.32 Therefore, a loss of CK5/6 expression may be a more reliable marker for distinguishing the small cell phenotype than a loss of p63 in the head and neck if p40 is unavailable.13 Unfortunately, IHC has limitations in detecting small cell carcinoma. For example, SqCC in its pure form is rarely positive for neuroendocrine markers; however, neuroendocrine markers lack sensitivity and specificity for the detection of small cell carcinoma.33,34 As a result, it is important to rely on the cytomorphologic findings and to thoroughly evaluate all the material for a patient with a possible oropharyngeal primary. This point is imperative because it is the presence of small cell carcinoma, and not the HPV status, that will drive the prognosis. The question of whether to perform HPV testing for these tumors at all is somewhat more complicated. Although HPV-related oropharyngeal SqCC demonstrates an improved prognosis over its HPV-negative counterparts, even in traditionally aggressive variants, HPV-related small cell carcinoma behaves as aggressively as HPV-negative small cell carcinomas.9,10 Because HPV testing for small cell carcinoma of the oropharynx fails to add prognostic information, recent guidelines from the College of American Pathologists do not recommend routinely testing these cancers for HPV.8 However, there are some compelling reasons to perform HR HPV–specific testing for these cases. The first reason for HPV testing is to identify the primary site of the malignancy; this consideration is especially applicable to FNA sampling of metastases. Small cell carcinoma can arise from a wide range of primary locations and can present with widespread metastases. In fact, HPV detection is one of the few tools available that can be used to identify the primary site of small cell carcinoma, with an oropharyngeal origin favored. Second, HPV testing may allow for a better understanding of this unique tumor and what it Cancer Cytopathology Month 2018


Figure 1. (A) Low-power view of a highly cellular smear composed of sheets of blue small round cells (Diff-Quik, ×10). (B) Characteristic features of small cell carcinoma, including nuclear molding, apoptotic bodies, and mitoses, are evident in this smear (Diff-Quik, ×40). (C) The cells of small cell carcinoma are fragile and often show a crush artifact as a result of smearing (Diff-Quik, ×40). (D) Highly cellular smear composed of aggregates and single blue small round cells (Papanicolaou, ×20). (E) Small aggregates and single cells with salt and pepper powdery chromatin in a background of scattered apoptotic bodies and necrotic debris (Papanicolaou, ×40). (F) Fragment of neoplastic cells showing molding, apoptotic bodies, and tumor necrosis (Papanicolaou, ×40).

Figure 2. (A,B) Core biopsy containing cells with both squamous and neuroendocrine differentiation (H & E, ×20). A p40 stain highlights the nuclei of the squamous component (p40, ×20). (C) Densely cellular fragment composed of blue small round cells with nuclear molding and a crush artifact (H & E, ×20). (D) A CD56 stain shows membranous staining of the small cell carcinoma component (CD56, ×20). (E) A synaptophysin stain shows patchy cytoplasmic labeling (synaptophysin, ×20). (F) HR HPV RNA ISH shows intense punctate cytoplasmic and nuclear staining, which indicates transcriptionally active virus in the malignant cells (HR HPV RNA ISH, ×20). HPV indicates human papillomavirus; HR, high-risk; ISH, in situ hybridization.


7

Original Article

means for patients. Although HPV-related SqCC of the oropharynx is increasing in incidence, HPV-related small cell carcinomas have not been identified as frequently as originally projected, perhaps because of a lack of HPV testing or awareness of this entity.12,13 Identification of this entity will allow for a better understanding of its epidemiology, clinical behavior, biomarkers, and potential treatment modalities. If HPV testing is performed for small cell carcinoma, however, the need for using HPV-specific testing is clear. Not only is IHC for p16 currently recommended for screening for HR HPV in all oropharyngeal SqCC, it is considered to be a sufficiently specific surrogate marker for standalone confirmation of the HPV status at this site.8 Because these tumors often metastasize to level II and III lymph nodes, p16 has also been suggested by expert consensus to also be specific for HPV positivity in nonkeratinizing SqCC metastases to these 2 lymph node levels.8 Unfortunately, however, small cell carcinomas often show p16 expression because of HPV-independent dysregulation of the Rb pathway, regardless of the primary location.35,36 As such, p16 is not a specific marker for HPV-related small cell carcinoma, and the only way to determine the true HPV status in a small cell carcinoma is to perform HR HPV–specific testing. There are several forms of HPV-specific technologies available for both liquid-based and formalin-fixed, paraffin-embedded samples that detect either DNA or RNA. Liquid-based samples rely primarily on polymerase chain reaction for the detection of HR HPV DNA or reverse transcription–polymerase chain reaction for E6/E7 RNA detection. For formalin-fixed, paraffin-embedded tissue, ISH for HR HPV DNA and ISH for HPV E6/E7 RNA are the most commonly used technologies. RNA detection is preferable to DNA detection because it requires an integrated, transcriptionally active HR HPV infection, and this eliminates a concern for a non-oncogenic infection. Furthermore, HR HPV RNA ISH has been shown to be more sensitive and specific than the HR HPV DNA ISH testing.30 As a result, HR HPV RNA ISH is considered to be the first-line platform for HR HPV–specific testing. In conclusion, HPV-related small cell carcinoma of the oropharynx is a unique HPV-related malignancy that is associated with distant metastases and a poor prognosis. The identification of a small cell component can be reliably performed with cytology preparations and is 8

crucial because this (and not the HPV status) determines the prognosis. As a result, every effort must be made to not overlook a small cell component in a sample from a suspected oropharyngeal metastasis. Furthermore, these cases may deserve a specific note in the report to be certain that the clinical team does not misinterpret the HPV status as a positive prognostic marker. However, HR HPV–specific testing can be clinically useful for identifying the primary site of the malignancy and for identifying cases to gain a better understanding of this entity, which we are only beginning to recognize. If HPV testing is performed on these tumors, p16 is not a useful marker for determining the HPV status because of its frequent expression in small cell carcinomas. Therefore, HR HPV–specific testing, ideally using the HR HPV RNA ISH assay, is required to make this diagnosis. FUNDING SUPPORT No specific funding was disclosed.

CONFLICT OF INTEREST DISCLOSURES The authors made no disclosures.

AUTHOR CONTRIBUTIONS Derek B. Allison: Data curation, formal analysis, investigation, methodology, project administration, writing–original draft, and writing–review and editing. Lisa M. Rooper: Conceptualization, data curation, methodology, and writing–review and editing. Sara Mustafa: Data curation, formal analysis, visualization, and writing–review and editing. Zahra Maleki: Visualization and writing–review and editing. Paul E. Wakely, Jr: Data curation, formal analysis, investigation, visualization, and writing–review and editing. Syed Z. Ali: Conceptualization, investigation, methodology, project administration, and writing–review and editing.

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23. Jalaly JB, Lewis JS, Collins BT, et al. Correlation of p16 immunohistochemistry in FNA biopsies with corresponding tissue specimens in HPV-related squamous cell carcinomas of the oropharynx. Cancer Cytopathol. 2015;123:723-731. 24. Othman MO, Abdelfatah MM, Padilla O, et al. The cellularity yield of three different 22-gauge endoscopic ultrasound fine needle aspiration needles. Diagn Cytopathol. 2017;45:426-432. 25. Allison DB, Wakely PE Jr, Siddiqui MT, Ali SZ. Nodular fasciitis: a frequent diagnostic pitfall on fine-needle aspiration. Cancer Cytopathol. 2017;125:20-29. 26. Lewis JS, Beadle B, Bishop JA, et al. Human papillomavirus testing in head and neck carcinomas: guideline from the College of American Pathologists. Arch Pathol Lab Med. 2017;142:559-597. 27. Holmes BJ, Maleki Z, Westra WH. The fidelity of p16 staining as a surrogate marker of human papillomavirus (HPV) status in fine needle aspirates and core biopsies of neck node metastases: implications for HPV testing protocols. Acta Cytol. 2015;59:97-103. 28. Xu B, Ghossein R, Lane J, Lin O, Katabi N. The utility of p16 immunostaining in fine needle aspiration in p16-positive head and neck squamous cell carcinoma. Hum Pathol. 2016;54:193-200. 29. Bishop JA, Ma XJ, Wang H, et al. Detection of transcriptionally active high-risk HPV in patients with head and neck squamous cell carcinoma as visualized by a novel E6/E7 mRNA in situ hybridization method. Am J Surg Pathol. 2012;36:1874-1882. 30. Rooper LM, Gandhi M, Bishop JA, Westra WH. RNA in-situ hybridization is a practical and effective method for determining HPV status of oropharyngeal squamous cell carcinoma including discordant cases that are p16 positive by immunohistochemistry but HPV negative by DNA in-situ hybridization. Oral Oncol. 2016;55: 11-16. 31. Ho WJ, Rooper L, Sagorsky S, Kang H. A robust response to combination immune checkpoint inhibitor therapy in HPV-related small cell cancer: a case report. J Immunother Cancer. 2018;6:33. 32. Serrano MF, El-Mofty SK, Gnepp DR, Lewis JS. Utility of high molecular weight cytokeratins, but not p63, in the differential diagnosis of neuroendocrine and basaloid carcinomas of the head and neck. Hum Pathol. 2008;39:591-598. 33. Lewis JS, Chernock RD, Bishop JA. Squamous and neuroendocrine specific immunohistochemical markers in head and neck squamous cell carcinoma: a tissue microarray study. Head Neck Pathol. 2018;12:62-70. 34. Rooper LM, Bishop JA, Westra WH. INSM1 is a sensitive and specific marker of neuroendocrine differentiation in head and neck tumors. Am J Surg Pathol. 2018;42:665-671. 35. Alos L, Hakim S, Larque AB, et al. p16 overexpression in highgrade neuroendocrine carcinomas of the head and neck: potential diagnostic pitfall with HPV-related carcinomas. Virchows Arch. 2016;469:277-284. 36. Yuan J, Knorr J, Altmannsberger M, et al. Expression of p16 and lack of pRB in primary small cell lung cancer. J Pathol. 1999;189:358-362.

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