The confirmation of human papillomavirus (HPV) as a causative agent for a subset of squamous cell carcinomas of the head and neck has resulted in a growing expectation for HPV testing in head and neck cancers. An increasing understanding of HPV-related tumorigenesis has informed this evaluation process in a manner that is moving wide scale, indiscriminant, and nonstandardized testing toward a more directed, clinically relevant, and standardized approach. This review addresses the current state of HPV detection and focuses on the importance, appropriate time, and need for HPV testing.
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Identification of human papillomavirus (HPV) in squamous cell carcinomas of the head and neck is rapidly becoming a means of tracking the presence and progress of disease relating to all aspects of patient care, including prognosis, tumor staging (ie, identifying site of tumor origin), and selection of patients who are most likely to benefit from tailored therapeutic options.
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At present, there is no standard approach for HPV testing of clinical samples. The challenge for the oncologic community is to implement standardized HPV testing using a method that is highly accurate, technically feasible, and cost effective.
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The use of p16 immunohistochemical staining is acceptable as a method of HPV detection, provided it is used and interpreted in a defined context that takes into account certain anatomic factors, histologic findings, and staining characteristics.
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The development of detection assays that are optimized for cytologic samples will lead to more widespread implementation of HPV testing and may obviate tissue acquisition and processing.
Introduction
Squamous cell carcinoma of the head and neck (HNSCC) has long been regarded as a monotonous disease entity. Important distinctions between anatomic subsites and natural histories have largely been ignored, given the histopathologic uniformity and response to treatment. Recent studies suggest considerable differences between some HNSCCs that go beyond variations related to tumor subsite and stage. In particular, a subset of HNSCCs, which is associated with the human papillomavirus (HPV), has emerged as a form of HNSCC with an epidemiologic, demographic, histopathologic, and clinical profile that deviates from the profile of conventional non-HPV–related HNSCC. HPV-associated cancers tend to occur more frequently in younger men, and tobacco smoking does not seem to be a strong cofactor in the development of these tumors. These cancers most frequently occur in the oropharynx, tend to exhibit a nonkeratinizing basaloid morphology, and are associated with improved clinical outcomes. In effect, recognition of this association with HPV amounts to the identification of a new and distinct disease entity.
Why is HPV testing important?
Until recently, clinicians have not been able to rely on prognostic markers other than tumor staging in their care of patients with HNSCC. Although numerous studies have addressed the prognostic relevance of cell proliferation (eg, Ki67, proliferating cell nuclear antigen), p53 immunohistochemical staining, apoptosis, aneuploidy, epidermal growth factor receptor overexpression, and various other markers of biological activity, none of these markers have proved consistently reliable across multiple studies. None of these markers are currently used as a routine part of pathologic evaluation for patient care. The absence of reliable prognostic markers has been offset to some degree by HPV testing. The detection of HPV in HNSCCs has recently emerged as a powerful biomarker, indicating a more favorable clinical outcome. Patients with HPV-positive tumors have a lower risk of tumor progression and death than patients with HPV-negative tumors. Accordingly, inclusion of HPV status as a parameter for emerging molecular staging systems is compelling, and routine HPV assessment will soon become part of the standard pathologic evaluation of all oropharyngeal carcinomas. Both the College of American Pathologists and the American Joint Committee on Cancer have recently recommended routine HPV testing as part of the standard pathologic evaluation of resected oropharyngeal squamous cell carcinomas for the purpose of molecular tumor staging.
The value of HPV testing is by no means confined to mere prognostication in patients with HNSCC. A study that analyzed the motivations for HPV testing in the clinical arena found that HPV testing was often initiated by the pathologist to help resolve difficult diagnostic dilemmas. For example, HPV testing of cervical lymph node metastases is a highly effective strategy for localizing the site of origin in those patients who present with neck metastases in the absence of an obvious primary tumor. In these patients, the detection of HPV in a lymph node metastasis is a reliable predictor of oropharyngeal origin ( Fig. 1 ). Similarly, HPV status can be used to clarify second primary or metastatic disease in those patients with HNSCC who subsequently develop squamous cell carcinoma in their lungs. In some instances, HPV status can inform the differential diagnosis, such as squamous-lined cysts of the lateral neck, where an HPV-positive cystic metastasis can easily be confused with an inflamed branchial cleft cyst.
As more is understood of the unique natural history of HPV-positive HNSCC, from viral infection to viral persistence to viral-induced malignant transformation, applications for HPV testing will undoubtedly continue to increase. The detection of HPV is emerging as a valid biomarker for discerning the presence and progress of disease, encompassing all aspects of patient care:
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Early cancer detection.
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More accurate tumor staging.
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Selection of patients most likely to benefit from specific treatments.
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Posttreatment tumor surveillance.
As for clinical research, study design and data analysis must incorporate HPV status into the next generation of clinical trials. Knowledge of HPV status is compulsory for meaningful comparison of treatment responses in patients enrolled in these trials. The direction of current clinical trials in which patient selection for specific therapies is predicated based on HPV tumor status dramatically heightens the stakes for accurate HPV detection.
Why is HPV testing important?
Until recently, clinicians have not been able to rely on prognostic markers other than tumor staging in their care of patients with HNSCC. Although numerous studies have addressed the prognostic relevance of cell proliferation (eg, Ki67, proliferating cell nuclear antigen), p53 immunohistochemical staining, apoptosis, aneuploidy, epidermal growth factor receptor overexpression, and various other markers of biological activity, none of these markers have proved consistently reliable across multiple studies. None of these markers are currently used as a routine part of pathologic evaluation for patient care. The absence of reliable prognostic markers has been offset to some degree by HPV testing. The detection of HPV in HNSCCs has recently emerged as a powerful biomarker, indicating a more favorable clinical outcome. Patients with HPV-positive tumors have a lower risk of tumor progression and death than patients with HPV-negative tumors. Accordingly, inclusion of HPV status as a parameter for emerging molecular staging systems is compelling, and routine HPV assessment will soon become part of the standard pathologic evaluation of all oropharyngeal carcinomas. Both the College of American Pathologists and the American Joint Committee on Cancer have recently recommended routine HPV testing as part of the standard pathologic evaluation of resected oropharyngeal squamous cell carcinomas for the purpose of molecular tumor staging.
The value of HPV testing is by no means confined to mere prognostication in patients with HNSCC. A study that analyzed the motivations for HPV testing in the clinical arena found that HPV testing was often initiated by the pathologist to help resolve difficult diagnostic dilemmas. For example, HPV testing of cervical lymph node metastases is a highly effective strategy for localizing the site of origin in those patients who present with neck metastases in the absence of an obvious primary tumor. In these patients, the detection of HPV in a lymph node metastasis is a reliable predictor of oropharyngeal origin ( Fig. 1 ). Similarly, HPV status can be used to clarify second primary or metastatic disease in those patients with HNSCC who subsequently develop squamous cell carcinoma in their lungs. In some instances, HPV status can inform the differential diagnosis, such as squamous-lined cysts of the lateral neck, where an HPV-positive cystic metastasis can easily be confused with an inflamed branchial cleft cyst.
As more is understood of the unique natural history of HPV-positive HNSCC, from viral infection to viral persistence to viral-induced malignant transformation, applications for HPV testing will undoubtedly continue to increase. The detection of HPV is emerging as a valid biomarker for discerning the presence and progress of disease, encompassing all aspects of patient care:
- •
Early cancer detection.
- •
More accurate tumor staging.
- •
Selection of patients most likely to benefit from specific treatments.
- •
Posttreatment tumor surveillance.
As for clinical research, study design and data analysis must incorporate HPV status into the next generation of clinical trials. Knowledge of HPV status is compulsory for meaningful comparison of treatment responses in patients enrolled in these trials. The direction of current clinical trials in which patient selection for specific therapies is predicated based on HPV tumor status dramatically heightens the stakes for accurate HPV detection.
When is HPV testing appropriate?
Site-Directed HPV Testing
HPV is not evenly distributed across all anatomic subsites in HNSCC. Instead, HPV infection is strongly correlated with oropharyngeal location, particularly the palatine and lingual tonsils. This correlation is so strong that the detection of HPV in an adjacent oropharyngeal site raises consideration of direct local extension from an oropharyngeal primary carcinoma. This preferential targeting likely reflects complex biological interactions between HPV and the highly specialized lymphoepithelial lining of the tonsillar crypts. Based on the localization of HPV-related HNSCC to the oropharynx, directives for routine HPV testing are generally restricted to those carcinomas arising from this specific anatomic subsite. Although HPV positivity is sometimes reported in HNSCCs arising outside the oropharynx, such as the sinonasal tract, expanding the scope of routine HPV testing is not warranted until well-designed studies establish a clear relationship between HPV infection at these nonoropharyngeal sites and a distinct natural history, including treatment responses.
HPV Testing of Lymph Node Metastases
In malignant transformation of the tonsillar epithelium, HPV does not act through a “hit-and-run” mechanism whereby its role is transient and limited to the initiation of tumorigenesis. Instead, the presence of HPV persists, and it is just as readily detected in metastatic implants as in the corresponding primary cancers. Consequently, a lymph node metastasis is quite suitable as a substrate for HPV testing, obviating additional tissue acquisition in those patients with small or even occult primary cancers.
How is HPV detected in clinical samples?
A Summary of the Biology of HPV as an Oncologic Agent
Various strategies are currently available for HPV analysis. An objective evaluation of these assays, including a comparison of their strengths and weaknesses, requires a fundamental understanding of the way HPV induces malignant transformation of the tonsillar epithelium, particularly its interaction with key components of the retinoblastoma (Rb) tumor suppressor gene pathway.
The p16 tumor suppressor gene, located on chromosome 9p21, is a member of the INK4 class of cell cycle inhibitors and represents a key component of the Rb pathway. Binding of the p16 tumor suppressor gene product with cyclin-dependent kinases 4 and 6 blocks its interaction with D-type cyclins, maintains the Rb gene in a hypophosphorylated state that binds the E2F transcription factor, and, in turn, prevents cell-cycle progression. In conventional (ie, non-HPV related) HNSCC, the p16 tumor suppressor gene is inactivated by various genetic and nongenetic (eg, promoter hypermethylation) modifications, such that the expression of its protein product is lost or dramatically diminished. By contrast, integration of high-risk HPV into the host genome is associated with upregulation of the p16 tumor suppressor gene product. Integration of HPV results in the deletion of the viral E2 gene promoter, causing transcription of E6 and E7. Binding of the E7 oncoprotein to the Rb protein leads to Rb protein degradation and, presumably, the compensatory overexpression of both cytoplasmic and nuclear p16 proteins in HPV-infected tumor cells.
Given the capacity to target and disrupt the Rb tumor suppressor gene pathway, HPV detection strategies focus on the detection of:
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The actual presence of HPV DNA
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Postintegration transcription of viral E6 and/or E7 messenger RNA (mRNA)
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The viral oncoproteins E6 and E7
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Altered expression of cellular proteins, such as overexpression of the p16 protein.
The Morphology of HPV-Related HNSCC
HPV-related HNSCC has a consistent microscopic appearance that diverges from the typical morphology of non-HPV–related HNSCC ( Table 1 ). Although the documentation of HPV status requires HPV testing, microscopic findings can successfully guide the implementation and interpretation of HPV analysis. Unlike most conventional HNSCCs, HPV-related HNSCCs do not typically arise from the surface epithelium showing a background of dysplastic changes, do not infiltrate as irregular cords and nests of cells that elicit a prominent desmoplastic stromal reaction, and do not show prominent cytoplasmic keratinization. Instead, HPV-related HNSCCs characteristically arise from the specialized reticulated epithelium of tonsillar crypts in the absence of dysplastic (ie, premalignant) changes in the surface squamous epithelium, often grow as rounded nests and lobules, are often permeated by infiltrating lymphocytes, and have tumor cells that display a high nuclear to cytoplasmic ratio, minimal keratinization, and a highly immature or basaloid appearance ( Fig. 2 ). The likelihood that the tumor is HPV related is very high when these features are present and well developed in oropharyngeal carcinoma.
| Histologic Features | Conventional Squamous Cell Carcinoma | HPV-Related Oropharyngeal Squamous Cell Carcinoma |
|---|---|---|
| Origin | Surface epithelium | Reticulated epithelium of tonsillar crypts |
| Surface dysplasia | Present | Absent |
| Growth pattern | Irregular cords and nests | Sheets and rounded lobules |
| Desmoplasia | Prominent | Often absent |
| Keratinization | Prominent | Minimal or absent |
| Differentiation | Moderately differentiated | Immature and basaloid (often interpreted as poorly differentiated) |
| Tumor-infiltrating lymphocytes | Usually absent | Often present |
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