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Context Human papillomavirus (HPV) is a major cause of oropharyngeal squamous cell carcinomas, and HPV (and/or surrogate marker p16) status has emerged as a prognostic marker that significantly impacts clinical management. There is no current consensus on when to test oropharyngeal squamous cell carcinomas for HPV/p16 or on which tests to choose. Objective To develop evidence-based recommendations for the testing, application, interpretation, and reporting of HPV and surrogate marker tests in head and neck carcinomas. Design The College of American Pathologists convened a panel of experts in head and neck and molecular pathology, as well as surgical, medical, and radiation oncology, to develop recommendations. A systematic review of the literature was conducted to address 6 key questions. Final recommendations were derived from strength of evidence, open comment period feedback, and expert panel consensus. Results The major recommendations include (1) testing newly diagnosed oropharyngeal squamous cell carcinoma patients for high-risk HPV, either from the primary tumor or from cervical nodal metastases, using p16 immunohistochemistry with a 70% nuclear and cytoplasmic staining cutoff, and (2) not routinely testing nonsquamous oropharyngeal carcinomas or nonoropharyngeal carcinomas for HPV. Pathologists are to report tumors as HPV positive or p16 positive. Guidelines are provided for testing cytologic samples and handling of locoregional and distant recurrence specimens. Conclusions Based on the systematic review and on expert panel consensus, high-risk HPV testing is recommended for all new oropharyngeal squamous cell carcinoma patients, but not routinely recommended for other head and neck carcinomas.

DEK::AFF2 carcinoma of the sinonasal tract is an emerging entity. The tumor is typically characterized by papillary proliferation of non-keratinizing squamous epithelial cells with monotonous cytologic features, which may mimic other sinonasal tumors. The confirmation of this gene fusion has thus far relied solely on next-generation sequencing, fluorescence in situ hybridization (FISH), or reverse transcription polymerase chain reaction (RT-PCR). This current study aimed to validate an immunohistochemical assay for AFF2 C-terminus as an ancillary marker. We first analyzed publicly available RNA sequencing data of sinonasal tumors from the national center for biotechnology information (NCBI) sequence read archive and identified 3 DEK::AFF2 carcinomas out of 28 sinonasal tumors. The gene expression of AFF2 was significantly higher in the fusion-positive cases compared to the wild-type tumors (p < 0.001), while DEK was not. We then optimized an immunohistochemical assay with an anti-AFF2 C-terminus antibody for ancillary diagnosis. Seventeen DEK::AFF2 carcinomas, including 11 cases with predominantly low-grade morphology and one showing glandular differentiation, as well as 78 DEK FISH-negative sinonasal tumors were evaluated by AFF2 immunohistochemistry (IHC). Sixteen of the 17 DEK::AFF2 carcinomas showed nuclear AFF2 expression in ≥30% of tumor cells, including one decalcified case that failed FISH and RT-PCR confirmation. The one case that was negative for AFF2 IHC in the tumor cells also lacked expression in the internal positive control. It was thus considered a failure of the IHC rather than a truly negative case and was excluded from the statistical analysis. All DEK FISH-negative sinonasal tumors were negative for nuclear AFF2 expression. The nuclear expression of AFF2 IHC showed 100% sensitivity and specificity for DEK::AFF2 carcinoma. Accordingly, AFF2 IHC is a highly sensitive and specific ancillary marker that distinguishes DEK-AFF2 carcinoma from the other sinonasal tumors with overlapping morphological features and may be an especially useful alternative for decalcified specimens.

Morphologic assessment is one of the most basic tools that pathologists use to classify tumors. Human papillomavirus (HPV)-related squamous cell carcinoma of the oropharynx has unique morphologic features that can be readily recognized under the microscope. Yet, these features are not widely recognized or uniformly reported. In our practice, we group oropharyngeal squamous cell carcinomas into ‘nonkeratinizing’, ‘nonkeratinizing with maturation’, and ‘keratinizing’ histologic types. The ‘nonkeratinizing’ type has a very strong association with HPV, while the ‘keratinizing’ type has a weaker association with the virus. ‘Nonkeratinizing with maturation’ is intermediate but much more closely related to the ‘nonkeratinizing’ type. This classification system parallels that of sinonasal and nasopharyngeal squamous cell carcinomas where nonkeratinizing squamous cell carcinomas are widely recognized histologic variants. This review will discuss this classification system and its utility in routine clinical practice.

Recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy with a significant unmet need for enhancing immunotherapy response given current modest efficacy. Here, we perform an in vivo CRISPR screen in an HNSCC mouse model to identify immune evasion genes. We identify several regulators of immune checkpoint blockade (ICB) response, including the ubiquitin C-terminal hydrolase 5 (UCHL5). Loss of Uchl5 in tumors increases CD8 + T cell infiltration and improved ICB responses. Uchl5 deficiency attenuates extracellular matrix (ECM) production and epithelial-mesenchymal-transition (EMT) transcriptional programs, which contribute to stromal desmoplasia, a histologic finding we describe as associated with reduced anti-PD1 response in human HNSCCs. COL17A1, a collagen highly and specifically expressed in HNSCC, mediates in part Uchl5 -mediated immune evasion. Our findings suggest an unappreciated role for UCHL5 in promoting EMT in HNSCC and highlight ECM modulation as a strategy to improve immunotherapy responses. UCHL5 is a deubiquitinating enzyme that cleaves Lys-48-linked polyubiquitin chains. Here, the authors discover through in-vivo CRISPR-Cas9 screens that Uchl5 is involved in immune evasion and modulation of extracellular matrix deposition in head and neck squamous cell carcinoma.

Although a strong etiologic relationship between human papillomavirus (HPV) and a majority of oropharyngeal squamous cell carcinomas has been established, the role of HPV in non-oropharyngeal head and neck carcinomas is much less clear. Here, we investigated the prevalence and clinicopathologic significance of HPV and its reported biomarkers, CDKN2A(p16) and CDKN1A(p21), in laryngeal squamous cell carcinomas in patients treated either with primary surgery and postoperative radiation or with definitive radiation-based therapy. Nearly all of 76 tumors were keratinizing and none displayed the nonkeratinizing morphology that is typically associated with HPV infection in the oropharynx. However, CDKN2A(p16) immunohistochemistry was positive in 21 cases (28%) and CDKN1A(p21) in 34 (45%). CDKN2A(p16) and CDKN1A(p21) status strongly correlated with each other ( P =0.0038). Yet, only four cases were HPV positive by DNA in situ hybridization or by reverse transcriptase PCR E6/E7 mRNA (all four were CDKN2A(p16) and CDKN1A(p21) positive). Unexpectedly, 9 additional tumors out of 20 CDKN2A(p16) positive cases harbored high-risk HPV DNA by PCR. For further investigation of this unexpected result, in situ hybridization for E6/E7 mRNA was performed on these nine cases and all were negative, confirming the absence of transcriptionally active virus. Patients with CDKN1A(p21)-positive tumors did have better overall survival (69% at 3 years) than those with CDKN1A(p21)-negative tumors (51% at 3 years) ( P =0.045). There was also a strong trend towards better overall survival in the CDKN2A(p16)-positive group ( P =0.058). Thus, it appears that the role of HPV is more complex in the larynx than in the oropharynx, and that CDKN2A(p16) and CDKN1A(p21) expression may not reflect HPV-driven tumors in most cases. Because of this, CDKN2A(p16) should not be used as a definitive surrogate marker of HPV-driven tumors in the larynx.

Metastatic carcinoma to cervical lymph nodes presenting as an unknown primary is quite common. In most cases, the primary site is ultimately identified. Carcinomas that remain of unknown primary after a thorough search are uncommon. This review will focus on those cases that initially present as unknown primaries, since this is the setting in which pathologists first encounter these cases and in which they play an important role in guiding patient management. Most are squamous cell carcinomas, the majority of which are human papillomavirus (HPV)-related and originate in the palatine tonsils and base of tongue. HPV-related oropharyngeal squamous cell carcinomas are increasing in incidence and have unique clinical and pathologic features that make them particularly likely to present as an unknown primary. Understanding these features has led to improved detection of the primary tumors. Further, even when the primary tumor is not found, prognosis is very dependent on characterization of the tumor HPV status. Papillary thyroid carcinomas may also initially present without a known or clinically detectable primary, either as a neck mass or incidentally in a neck dissection performed for another indication. The latter is a very indolent disease. Finally, primary salivary gland carcinomas may mimic an unknown primary and need to be distinguished from cutaneous metastases to the parotid gland, which may present without a recognized skin tumor. Here, we review the clinical and pathologic features of these entities and provide a systematic approach to their diagnosis.

Human papilloma virus (HPV) is an etiologic agent in a subset of oropharyngeal squamous cell carcinomas (SCCs). The aim of this study was to sub-classify SCC of the oropharynx based upon histologic features into nonkeratinizing (NK) SCC, keratinizing (K) SCC, and hybrid SCC, and determine the frequency of HPV and patient survival in each group. Patients with oropharyngeal SCC with a minimum of 2 years of clinical follow-up were identified from radiation oncology databases from 1997 to 2004. All patients received either up front surgery with postoperative radiation or definitive radiation based therapy. In situ hybridization (ISH) for high-risk HPV subtypes and immunohistochemistry for p16, a protein frequently up-regulated in HPV-associated carcinomas, were performed. Overall and disease-specific survival were assessed. Of 118 cases, 46.6% were NK SCC, 24.6% K SCC and 28.8% hybrid SCC. NK SCC occurred in slightly younger patients that were more often male. It more frequently presented with lymph node metastases and was surgically resected compared to K SCC. NK SCC was significantly more likely to be HPV and p16 positive than KSCC (P < 0.001) and to have better overall and disease-specific survival (P = 0.0002; P = 0.0142, respectively). Hybrid SCC was also more likely than K SCC to be HPV and p16 positive (P = 0.003; P = 0.002, respectively) and to have better overall survival (P = 0.0105). Sub-classification of oropharyngeal SCC by histologic type provides useful clinical information. NK SCC histology strongly predicts HPV-association and better patient survival compared to K SCC. Hybrid SCC appears to have an intermediate frequency of HPV-association and patient survival.

Salivary duct carcinoma (SDC) is a high-grade salivary gland malignancy with great morphological resemblance to invasive ductal carcinoma (IDC) of the breast. Rarely, female patients may have a past history of both SDC and IDC. When these patients present with distant metastasis, accurate identification of the primary tumor is particularly difficult. Additionally, rare metastasis of SDC to the breast and IDC to the salivary (parotid) gland can also present a diagnostic challenge. Our aim was to develop an immunohistochemical panel that reliably distinguishes SDC from IDC. We included all SDCs diagnosed from 1989 to 2016 (23 cases) and 29 treatment naïve and histologically similar IDCs. All cases were stained with androgen receptor (AR), estrogen receptor-alpha (ER-α), progesterone receptor (PR), HER-2, CK5/6, p63, and beta-catenin. The great majority (> 90%) of both SDCs and IDCs reacted positively to AR. The main discrepancy in the immunohistochemical profiles was a distinctly different reactivity to ER-α, PR and HER-2. While 28 IDCs (96.6%) reacted positively to ER-α and/or PR, the majority expressing both (82.8%) with a moderate to strong staining intensity, only 2 SDCs expressed ER-α (8.7%) and 5 others expressed PR (21.7%) with only one case expressing both (P value < 0.05). On the other hand, 8 SDC (34.8%) were positive for HER-2 while none of the IDCs were positive (P value < 0.05). ER-α, PR, and HER-2 may be helpful to distinguish SDC from IDC. Positive reactivity to ER-α, PR or both and negative HER-2 favors a diagnosis of IDC while ER-α, PR negative, HER-2 positive tumors are more likely SDC.

Poorly differentiated thyroid carcinomas (PDTC) in young individuals are rare and their clinical and histopathologic features, genetic mechanisms, and outcomes remain largely unknown. Here, we report a detailed characterization of a series of six PDTC in patients ≤21 years old defined by Turin diagnostic criteria studied for mutations and gene fusions characteristic of thyroid cancer using targeted next-generation sequencing (NGS) and whole-exome sequencing (WES). All tumors had solid, insular, or trabecular growth pattern and high mitotic rate, and five out of six tumors showed tumor necrosis. Targeted NGS assay identified somatic mutations in the DICER1 gene in five of six (83%) tumors, all of which were “hotspot” mutations encoding the metal-ion binding sites of the RNase IIIb domain of DICER1 . WES was performed in five cases which confirmed all hotspot mutations and detected two tumors with additional inactivating DICER1 alterations. Of these two, one was a germline pathogenic DICER1 variant and the other had loss of heterozygosity for DICER1 . No other mutations or gene fusions characteristic of adult well-differentiated thyroid cancer and PDTC ( BRAF , RAS , TERT , RET/PTC, and other) were detected. On follow-up, available for five patients, three patients died of disease 8–24 months after diagnosis, whereas two were alive with no disease. The results of our study demonstrate that childhood- and adolescent-onset PDTC are genetically distinct from adult-onset PDTC in that they are strongly associated with DICER1 mutations and may herald DICER1 syndrome in a minority. As such, all young persons with PDTC may benefit from genetic counseling. Furthermore, their clinically aggressive behavior contrasts sharply with the indolent nature of the great majority of thyroid tumors with DICER1 mutations reported to date.

In the 9 years since the last World Health Organization (WHO) classification for head and neck tumors, a great deal has changed. In particular, human papillomavirus (HPV) has emerged as the major etiologic agent and patient prognostic marker for squamous cell carcinoma, most profoundly in the oropharynx. It also casts a long shadow over all of the rest of head and neck cancer given its biologic and prognostic implications. By contrast, little has changed regarding our knowledge of Epstein–Barr virus in head and neck cancers, except as it relates to HPV in nonkeratinizing-type nasopharyngeal carcinomas. This article discusses some of the major advances in our understanding of virus-related squamous cell carcinoma of the head and neck and suggests several specific concepts and terminology for incorporation into the next WHO classification.