HPV and p16 Testing in Oropharyngeal Squamous Cell Carcinoma: Indication, Interpretation, and Implication [Hot Topic]

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Joaquín García, M.D.

Human papillomavirus (HPV) is regarded as a causative agent in the majority of oropharyngeal squamous cell carcinoma cases. For this reason, HPV and p16 testing is used to determine prognosis and treatment strategies for these patients. Dr. Joaquín García describes the various HPV and p16 testing platforms and respective performance characteristics as they relate to clinical management. More specifically, Dr. García discusses HPV DNA in situ hybridization (ISH), HPV RNA ISH, polymerase chain reaction (PCR), and p16 immunochemistry.

Presenter and Credentials:
Joaquín García, M.D., Consultant in the Division of Anatomic Pathology at Mayo Clinic in Rochester, Minnesota, Vice Chair of Laboratories, and Medical Director of the Histology Laboratory.

Transcript

Our speaker for this program is Dr. Joaquín García, a Consultant in the Division of Anatomic Pathology at Mayo Clinic in Rochester, Minnesota. Dr. Joaquín García is also the Vice Chair of Laboratories and Medical Director of the Histology LaboratoryWelcome to Mayo Medical Laboratories Hot Topics. These presentations provide short discussion of current topics and may be helpful to you in your practice. Today our topic is an update on the role of human papillomavirus and p16 in the pathogenesis of oropharyngeal squamous cell carcinoma and indications for and interpretation of testing.

Thank you for the introduction. It is an honor and a privilege to speak to you on a topic that promises to change the way we view the biology and management of head and neck cancer. There are over 400,000 cases of head and neck cancer globally each year; and they often impart a significant degree of morbidity as well as mortality.  About 90% of these cases are squamous cell carcinoma.  Advances in the field have dramatically improved function, organ preservation, quality of life, and survival.  Major developments include primary chemoradiotherapy for unresectable disease and the addition of chemotherapy to adjuvant radiotherapy.  Surgical techniques have also improved dramatically over the years. Despite these advances, 50% of patients will recur locally and 30% will develop distant metastases.

I have no disclosures.

This talk may be helpful as you consider the use of HPV and p16 testing in oropharyngeal squamous cell carcinoma, or OPSCC. Detect indications for HPV and p16 testing in OPSCC; interpret various clinical assays designed for HPV and p16 detection in OPSCC; and recognize implications of HPV and p16 testing results in OPSCC.

When clinicians and scientists discuss diseases driven by virus—HPV for example—we often conclude with impact on patient care.

Understanding the role of HPV in head and neck cancer—specifically oropharyngeal squamous cell carcinoma—requires that we also understand patient management strategies.  And, furthermore, where there is opportunity to improve.

In addition to discussing therapeutic options in the setting of oropharyngeal squamous cell carcinoma, we will also review key features such as epidemiology, histology, anatomy, HPV itself, and the strengths and limitations of clinical assays currently available.

Care.

Patients with oropharyngeal squamous cell carcinoma are often placed into 1 of 2 categories:  HPV-positive or HPV-negative.  In the oropharynx, it was originally thought that 40 to 50% of squamous cell carcinoma cases were HPV-positive; today that figure is much higher.  In 2010, 4,200 new OPSCC cases were attributed to alcohol and tobacco, while 8,400 were attributed to HPV.

HPV-positive patients have shown better overall survival and cure rates when compared to their HPV-negative counterparts. Because of this, consideration is given to de-escalation of therapy in HPV-positive cases.

Current standard of care is platinum-based chemoradiotherapy, which has been shown to improve overall survival, progression free survival, and locoregional control.  Surgery is often performed as salvage treatment.

Historically, oropharyngeal squamous cell carcinoma was treated with aggressive surgeries with long recovery periods and, not uncommonly, positive surgical margins.  More recently, transoral surgical techniques such as transoral CO2 laser microsurgery and transoral robotic surgery with the da Vinci surgical robot minimize disturbance of normal anatomic relationships and vascularity, thereby limiting the morbidity of surgical exposure.

In our practice at the Mayo Clinic, however, surgery is employed in a significant proportion of cases of oropharyngeal squamous cell carcinoma.

In some cases, patients may be given a higher dose of radiation at the primary tumor site if they are not considered surgical candidates.  Patients with oropharyngeal squamous cell carcinoma not uncommonly, receive neck irradiation as well.  While some institutions will irradiate all neck levels with the same dose, others will provide a boost at the level of node positivity or extracapsular extension.

Radiation exposure does not come without side effects.  In ECOG 2399, which used induction chemotherapy with carboplatin and paclitaxel followed by CRT with taxol and 70 Gy, 49% of patients had moderate to severe swallowing impairment after 3 months.  Other side effects include hypothyroidism, infection, scarring, challenges with speech, and even cancer.

Cisplatin is considered standard of care for patients with oropharyngeal squamous cell carcinoma in most cases.  Patients may receive cisplatin if there are positive margins, extracapsular extension, 2 or more positive lymph nodes, or a constellation of other findings that portend a worse prognosis.

As you would expect, adding chemotherapy to a treatment regimen does not come without risks.  Cisplatin, the agent most commonly used, can also aggravate the side effects of radiation. Clinicians and patients must worry about nephrotoxicity, neurotoxicity, ototoxicity, blood count abnormalities, and nausea.

Although HPV status does not currently impact surveillance strategies for patients with oropharyngeal squamous cell carcinoma that may not be true in the future.

Both CT and MRI provide clinical utility in determining anatomic location and extent of disease.  This collection of images highlights a left-sided tonsillar squamous cell carcinoma with Level IIa lymph node involvement.  The cystic architecture of the lymph node is not uncommon.

This is a CT-PET of a patient with a right-sided tongue base squamous cell carcinoma.  Again, you can see involvement of Level IIa lymph nodes.  One challenge associated with CT-PET is that Waldeyer’s ring is hypermetabolic in the normal state; this can potentially obscure the signal of an occult primary.

Patients.

The incidence of OPSCC is on the rise. The number of cigarette smokers has declined in recent years, mostly as a result of dramatic public health efforts and smoking cessation programs.  Naturally, one would expect the incidence of squamous cell carcinoma of the head and neck to decline as well.  It turns out that the incidence has not declined; in fact, the incidence has remained relatively unchanged and some studies suggest it is actually rising.  This observation is considered at least partially related to cases of HPV-positive oropharyngeal squamous cell carcinoma.

Like many cancers of the head and neck, oropharyngeal squamous cell carcinoma is seen predominantly in males.  The ratio is approximately 3:1.

For decades, head and neck squamous cell carcinoma has been largely attributed to long-term exposure to carcinogens such as alcohol, cigarettes, and betel nut.  More recently, we have witnessed a bimodal distribution of patients afflicted by OPSCC, with the earlier peak representing so-called HPV-associated cases. Patients are younger, lack a history of significant tobacco and/or alcohol exposure, and have often engaged in earlier and more frequent sexual contact.

Histology.

Oropharyngeal squamous cell carcinoma should be considered histologically similar but distinct from squamous cell carcinoma arising from other anatomic subsites . Waldeyer’s ring includes palatine and lingual tonsils of the oropharynx and pharyngeal and tubal tonsils of the nasopharynx.  The surface epithelium is often referred to as lymphoepithelium; the coexistence of both epithelial and lymphoid cells. This epithelium is reticulated; that is, there is an incomplete basement membrane that gives surface epithelium a serrated or irregular appearance.  Often times, rather than a thick basement membrane, what separates the lymphocyte from the pharyngeal lumen is nothing more than a layer of epithelial cytoplasm.

The inconsistent basement membrane challenges whether dysplasia or carcinoma in situ may exist in the oropharynx.  Another peculiar finding is that oropharyngeal primary tumors infrequently elicit a robust desmoplastic reaction within surrounding stroma.

Oropharyngeal primary squamous cell carcinoma can be categorically regarded as keratinizing or nonkeratinizing.  Whereas the majority of keratinizing tumors are HPV-negative, the majority of nonkeratinizing tumors are HPV-positive.

This pair of micrographs shows examples of keratinizing and nonkeratinizing squamous cell carcinoma of the oropharynx.

In addition to conventional squamous cell carcinoma, histomorphologic variants such as basaloid, papillary, and adenosquamous carcinoma are occasionally encountered.

Anatomy.

What is the oropharynx?  The current state of affairs in head and neck oncology requires that investigators, pathologists, surgeons, and oncologists alike understand precisely what is the oropharynx.  The oropharynx extends from the plane of the hard palate superiorly to the plane of the hyoid bone inferiorly.  It is distinguished from the oral cavity by the junction of the soft and hard palate superiorly and the line of the circumvallate papillae inferiorly.  The lateral borders of the oropharynx are the anterior pillars of the fauces.  In the world of oncology, there are 4 major constituents:  base of tongue, soft palate, palatine tonsils, and pharyngeal wall.

HPV.

Human papillomaviruses are encapsulated, nonenveloped, double-stranded DNA viruses that are members of the Papillomaviridae family.  This family has well over 100 subtypes, also known as genotypes. HPV can be isolated in approximately 25% of cases of head and neck squamous cell carcinoma.  Of all head and neck sites, HPV has the strongest etiopathogenic link to the oropharynx.

HPV genotypes have different profiles, including variable tissue trophism and biologic roles in human disease. HPV genotypes are placed into 1 of 2 categories: low-risk andhigh-risk.  The more common low-risk genotypes are 6 and 11 while the more common high-risk genotypes are 16, 18, 31, 33, and others.  Low-risk genotypes are frequently associated with benign neoplasms such as squamous papillomas; high-risk genotypes are more commonly associated with malignant neoplasms such as squamous cell carcinoma.

The HPV circular genome is usually around 8,000 base pairs in length and contains approximately 80 genes, which are categorized as early (E) or late (L).  The designations of early and late refer to the temporal sequence of gene expression.  It turns out that E6 and E7 oncoproteins are expressed in low- and high-risk genotypes; however, these proteins bind tumor suppressor proteins with greater affinity when expressed by high-risk genotypes.  Late genes, L1 and L2, are capsid proteins.

In the pathology literature, E6 and E7 receive the most attention by a landslide.

The E6 protein binds p53 and, in concert with E6-associated protein (E6AP), which is a cellular ubiquitin ligase, marks it for degradation by way of a proteosome-dependent pathway.  E6 is also thought to work outside of the p53 pathway.

The E7 protein, on the other hand, binds and inactivates proteins of the retinoblastoma gene family of tumor suppressors. Retinoblastoma will also modify the activity of other tumor suppressor proteins such as p16, a cyclin-dependent kinase inhibitor.

Testing.

HPV testing is primarily considered a prognostic tool as patients with HPV-positive oropharyngeal squamous cell carcinoma have exhibited better progression-free survival, decreased local-regional failure, and improved overall survival when compared to HPV-negative cohorts.

In addition to serving as strong prognostic tools, HPV in situ hybridization and p16 immunohistochemistry may assist in determining the origin of an unknown primary.  The most common scenario is a cervical lymph node or neck mass that shows morphologic and immunophenotypic features of metastatic squamous cell carcinoma.  Although positive testing in either of these assays is not exclusive to squamous cell carcinoma, such results strongly suggest the possibility of an oropharyngeal primary lesion.

Depending on the study, HPV can be detected in 50 to 80% of cases of oropharyngeal squamous cell carcinoma.  Each HPV detection method carries a different profile of clinical sensitivity and specificity.  Similarly, the ability to correlate test findings with a light microscope and accessibility with respect to resources also varies.

Most studies have found a strong correlation between p16 immunoreactivity and HPV positivity by polymerase chain reaction or in situ hybridization.  In other words, p16 can be overexpressed in the absence of HPV DNA or E6/E7 mRNA transcripts.  For this reason, the clinical specificity remains a point of challenge. p16 interpretation requires histologic correlation. Lastly, not all but a fair number of immunohistochemistry laboratories offer p16 immunostaining.

This pair of micrographs shows p16 immunohistochemistry in a cervical lymph node.

DNA in situ hybridization for HPV is often considered the gold standard for HPV detection.  However, it carries a low clinical sensitivity; there are enough false negatives that you should be inspired to question a negative result when the clinical and histologic parameters all seem to point towards an HPV-associated tumor.  Several tumors with HPV DNA and E6/E7 mRNA have been negative by DNA in situ hybridization.

The specificity of DNA in situ hybridization for HPV is remarkable as it employs probes for select sequences (that is, low- or high-risk genotypes).

Because this assay is interpreted using the light microscope, it not only allows for the detection of viral DNA, it provides some insight into the localization of HPV DNA as well.

DNA in situ hybridization is readily available in most large-scale reference laboratories and a fair number of laboratories belonging to institutions with smaller case volumes.

Importantly, DNA in situ hybridization gives no indication of biological activity.  That is, the presence of HPV DNA tells you little if anything about the role of HPV in the initiation and/or maintenance of malignant transformation.

This pair of micrographs shows HPV DNA in situ hybridization in a primary tumor.

Detecting E6/E7 mRNA transcripts provides exquisite sensitivity and specificity.  Moreover, this assay affords tremendous insight into the expression status of oncogenes E6 and E7.  The presence of E6 and E7 mRNA transcripts, for all intents and purposes, represents biologically active HPV DNA, regardless of localization.

This assay is interpreted using the light microscope.

The availability of this assay to clinical laboratories is currently limited but widening.

This pair of micrographs shows HPV RNA in situ hybridization in a primary tumor.

The best feature of the polymerase chain reaction in HPV detection is its high level of sensitivity.  PCR is capable of detecting an incredibly low volume of viral transcripts.

This assay is specific in that the primers may be designed for a unique sequence of interest—such as thesequence of particular genotype.  On the other hand, its sensitivity is so great that viral DNA from histologically normal mucosa or contaminants can potentially give rise to false-positive results.

Polymerase chain reaction results cannot be correlated with light microscopic evaluation.

Lastly, polymerase chain reaction is laborious and relatively few labs offer HPV detection as a clinical test.

This pair of micrographs shows polymerase chain reaction using universal HPV and HPV 16 primers.

In sum, HPV and p16 testing are important in oropharyngeal squamous cell carcinoma to identify patients that should have treatment de-escalated.  Cases of HPV-positive oropharyngeal squamous cell carcinoma are typically in young patients and the tumors are nonkeratinizing.  The oropharynx consists of the base of tongue, soft palate, palatine tonsils, and pharyngeal wall.  High-risk HPV genotypes can be identified using an array of clinical assays; knowing the performance characteristics of the assay you employ is instrumental to both patient care and communication with colleagues.

If you enjoyed this presentation, please join us in Rochester, Minnesota on April 13 to 14, 2015 for our Diagnostic Molecular Pathology conference. For more information, visit MayoMedicalLaboratories.com

Thank you for your attention.

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