HPV-related squamous cell carcinoma of the head and neck: An update on testing in routine pathology practice

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Abstract

Oropharyngeal squamous cell carcinoma caused by high-risk types of human papillomavirus (HPV) is now a well-recognized tumor entity whose incidence is on the rise. Most HPV-related oropharyngeal squamous cell carcinomas have a distinct histomorphology, and most patients fit a typical clinical profile. Importantly, HPV-related oropharyngeal carcinoma patients overall have significantly improved outcomes when compared to their HPV-negative counterparts, and the differences in tumor biology may soon lead to modifications in how they are treated. While high-risk HPV can be detected in a significant minority of head and neck squamous cell carcinomas across anatomic subsites in the head and neck, it has become clear in recent years that the biologically and clinically favorable features are limited to tumors that harbor transcriptionally active, high-risk HPV, something that occurs predominantly (but certainly not exclusively) in the oropharynx. It is now acknowledged that detecting transcriptionally active, high-risk HPV is a necessity in routine clinical practice, but there is considerable confusion among pathologists and clinicians alike about the subsites and settings in which HPV testing should be performed. Compounding this lack of clarity is the fact that there are multiple HPV testing options available, but currently there is no clear consensus on which test or combination of tests is optimal for routine diagnostic use. This review serves as an update for practicing pathologists on the current status of HPV (and surrogate marker) testing in head and neck cancers.

Section snippets

Why is HPV testing in head and neck cancer important?

Recent work has definitively established transcriptionally active HPV-related squamous cell carcinoma (HPV-SCC) as a distinct form of head and neck cancer with an increasing incidence.1, 2, 3, 4, 5 HPV-SCC differs from conventional HPV-negative SCC in significant ways, including its typical clinical profile: compared to conventional SCC, HPV-SCC tends to arise in younger patients with higher rates of high-risk sexual practices, more frequently in non-smokers, and with more frequent nodal

General considerations for HR-HPV testing

Despite the recognized importance of HPV testing, there is widespread confusion over when HPV testing should be performed on a head and neck cancer.18, 19, 20 The majority of head and neck HPV-SCCs arise in the oropharynx (i.e., tonsils and base of the tongue), where the reticulated tonsillar lymphoepithelium is thought to be particularly susceptible to infection by HPV and to associated tumorigenesis.21 Modern studies, particularly those in the United States, show that up to 80% of

Routine histology

Sometimes overlooked in discussions regarding HPV testing is the fact that most HPV-SCC exhibit a characteristic morphologic appearance on routine hematoxylin and eosin-stained sections.31 HPV-SCC arises from the tonsillar crypts and invades as rounded nests and sheets of cells that do not typically elicit a desmoplastic stromal reaction. The HPV-SCC tumor cells are highly reminiscent of the non-neoplastic tonsillar crypt epithelial cells, with indistinct cell borders, round to oval nuclei with

Polymerase chain reaction-based techniques

Polymerase chain reaction (PCR)-based techniques that utilize either general consensus or HPV type-specific primers have the ability to amplify and detect even a minute amount (e.g., less than one copy per cell) of HPV DNA. As a result, this HPV testing method is highly sensitive for the presence of HPV. However, simply finding HPV DNA cannot distinguish HPV infections that are truly causative of HPV-SCC (i.e., transcriptionally active) from those that are clinically insignificant (i.e.,

DNA in situ hybridization

DNA in situ hybridization (ISH) is a technique that allows for the visualization of DNA in the histologic context, thus confirming that any HPV DNA signal detected is present within the tumor cells. The finding of punctate nuclear signals in a clonally expanded population of tumor cells is highly specific for integration of viral DNA into the host genome (Fig. 1C). Accordingly, a significant advantage of DNA ISH is its high specificity. In addition, because it can be performed utilizing widely

RNA in situ hybridization

The recent development of ISH for detecting the HPV E6/E7 mRNA transcripts in formalin-fixed paraffin-embedded tumor tissues is an exciting advance in HR-HPV testing (Fig. 1D). Because it is detected in the histologic context, RNA ISH has the same advantage of high specificity as does DNA ISH. It has the added benefit of identifying not only the presence of HR-HPV but also HR-HPV in transcriptionally active form.25, 39, 40 With early experience, the sensitivity and specificity of RNA ISH appear

p16 Immunohistochemistry

Perhaps, the simplest and least expensive HPV testing type is the detection of a surrogate marker of HR-HPV infection, p16. p16 is a tumor suppressor protein that inhibits CDK4- and CDK6-dependent phosphorylation of the retinoblastoma protein required for cell cycle progression. Loss of p16 expression is a common event in HPV-negative head and neck SCC through homozygous deletion, promoter methylation, and inactivating mutations.41 The rationale for p16 testing as a surrogate for

Multimodality testing

An increasing number of centers are employing more than one HPV testing approach for oropharyngeal SCCs.22, 36, 45 The most common multimodality approach utilizes p16 immunohistochemistry in addition to either a DNA PCR or ISH-based method. These tests can be done simultaneously, or in a stepwise, algorithmic fashion, where p16 is used as a screening test (due to very high sensitivity), followed by a HPV-specific test.22, 36 An advantage of these approaches is that they combine the high

HPV testing on fine needle aspiration material

Because of the tendency of HPV-SCC to metastasize to cervical lymph nodes, the first pathologic material available for HR-HPV testing is often a fine needle aspiration (FNA), which is used for the initial evaluation and diagnosis of metastatic HPV-SCC (Fig. 2).51, 52 In some cases, the FNA material may be the only tissue ever available to test.53 As a result, accurate and efficient testing methods for HR-HPV are needed for the evaluation of head and neck SCCs in cytologic preparations. Standard

Conclusions

While the importance of HR-HPV testing in oropharyngeal SCCs is clear, there remains considerable confusion among clinicians and pathologists regarding when and how HPV testing should be performed in head and neck pathology. At this time, it is recommended that HPV testing on head and neck cancers should be limited to assays for high-risk HPV types, and it should be routinely performed on (but also limited to) oropharyngeal carcinomas and metastatic SCCs in neck lymph nodes from unknown primary

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