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HPV and p16 Testing in Oropharyngeal Squamous Cell Carcinoma

Methodology, Interpretation, and Significance

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Published: March 2012

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Human papillomavirus (HPV) is detected in 50% to 80% of oropharyngeal squamous cell carcinomas (OPSCCs). Dr. García describes the various testing methods for HPV detection, including DNA in situ hybridization (ISH), polymerase chain reaction (PCR), p16 immunochemistry, and E6/E7 messenger ribonucleic acid (mRNA). He also discusses the interpretation and significance of the assay results.

Presenter: Joaquín García, MD

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Transcript

Introduction

Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussions of current topics and may be helpful to you in your practice.

Our presenter for this program is Dr. Joaquín García from the Division of Anatomic Pathology in the Department of Laboratory Medicine and Pathology at Mayo Clinic in Rochester, Minnesota. Dr. García will describe the role of human papillomavirus and p16 in the pathogenesis of oropharyngeal squamous cell carcinoma and discuss indications for and interpretation of testing for HPV and p16. Thank you Dr. García.

Thank you for the kind 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 in the coming years. My intent today is to package the current understanding of HPV and p16 testing, interpretation, and significance in the context of oropharyngeal squamous cell carcinoma in a useable way for head and neck practitioners; that is, laboratory technicians, investigators, pathologists, surgeons, and oncologists.

Objectives

I have 4 primary objectives today: the first is to identify the key principles and events related to HPV and p16 in the pathogenesis of oropharyngeal squamous cell carcinoma, or OPSCC; the second is to highlight indications for HPV and p16 testing in oropharyngeal squamous cell carcinoma; third, we will spend time discussing the interpretation of the most commonly used clinical assays designed for HPV and p16 detection in oropharyngeal squamous cell carcinoma; and lastly, I will call attention to the strengths and limitations of current assays available for HPV and p16 testing in oropharyngeal squamous cell carcinoma.

Outline

I will attempt to look at this issue with a wide angle lens, focusing on principles and phenomena rather than inundating you with a deluge of references and statistics. Although I will certainly mention some of the more important studies and statistics, anyone driven to learn more about this topic is strongly encouraged to browse the references listed at the conclusion of this presentation.

To this end, we will start with a review of oropharyngeal squamous cell carcinoma - followed by a discussion regarding our current understanding of HPV’s and p16’s role in oropharyngeal squamous cell carcinoma. We will then transition into indications for HPV and p16 testing in oropharyngeal squamous cell carcinoma. At least equally as important, we will spend time discussing the idiosyncrasies of HPV and p16 test interpretation in oropharyngeal squamous cell carcinoma. Finally, we will wrap up with a review of the salient features of HPV and p16 testing in this context.

Oropharyngeal Squamous Cell Carcinoma in Review

Oropharyngeal Squamous Cell Carcinoma in Review.

Oropharynx Anatomy

HPV can be detected in a number of anatomic subsites within the head and neck; in normal, reactive, and neoplastic conditions. As you well know, HPV has been detected in several tumor types other than squamous cell carcinoma. I mention this to point out the ubiquitous nature of HPV in the head and neck, not to draw parallels between the oncogenic role of HPV in squamous cell carcinoma and other head and neck malignancies.

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. To this end, 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. Most importantly, the oropharynx includes the base of tongue and bilateral palatine tonsils.

Epidemiology of OPSCC — Demographics

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, there has been a paradigm shift in how we think about oncogenesis in squamous cell carcinoma of the head and neck; specifically, in the oropharynx. We have recently witnessed a bimodal distribution of patients afflicted by squamous cell carcinoma of the oropharynx. It turns out that there is also a new segment of the population that is being confronted with squamous cell carcinoma with increased regularity. These patients are the so-called HPV-associated cases of oropharyngeal squamous cell carcinoma. Patients are typically younger, often between 30-40 years of age, lack a history of significant tobacco and/or alcohol exposure, and have engaged in "high-risk" sexual behavior. This latter term can be somewhat nebulous so allow me to be explicit: orogenital sexual practices.

Epidemiology of OPSCC — Incidence

Think about this: the number of cigarette smokers has actually declined in recent years, mostly as a result of dramatic public health efforts and smoking cessation programs. Naturally, one would initially 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 that the incidence is actually rising. This phenomenon is considered partly to be a function of the new cohort of patients afflicted by oropharyngeal squamous cell carcinoma.

Management Strategies in OPSCC

Primary chemoradiation is considered the standard of care nationally, with surgery often performed as salvage treatment. In our practice at the Mayo Clinic, however, surgery is the treatment mainstay for most cases of oropharyngeal squamous cell carcinoma. Clear margins, as in many settings of head and neck cancer, is the primary goal so frozen section to evaluate clearance is not uncommon.

Clinical stage 1 and 2 patients (ie patients with small tumors and no lymph node involvement) are often treated with surgery alone.

Clinical stage 3 and 4 patients almost invariably receive radiation. The side effects of radiation are worth considering; fibrosis and scarring may lead to swallowing difficulty, hypothyroidism, and neck pain.

Chemotherapy is reserved for patients with positive surgical margins or extranodal spread. As you would expect, adding chemotherapy to a treatment regimen may come at the expense of adverse events. Cisplatin, the agent most commonly used, can aggravate the side effects of radiation. It may cause nausea, vomiting, and decreased blood counts. The long-term effects of Cisplatin are renal dysfunction, neuropathy, and ototoxicity.

In general, patients are considered to have a significant improvement in survival when treated with post-operative adjuvant therapy. Concurrent chemoradiotherapy has traditionally been considered to offer a better survival benefit when compared to radiotherapy alone. However, recent studies have argued that, in the setting of HPV-associated oropharyngeal squamous cell carcinoma, there is no benefit to adding chemotherapy to the post-operative treatment regimen. This is not an unsettled issue, however, and is certainly something to keep an eye on.

Currently, at the Mayo Clinic, HPV-positive and negative patients are treated essentially the same. However, considerations are being given to deescalating treatment intensity given the less aggressive behavior of HPV-associated oropharyngeal squamous cell carcinoma.

In terms of targeted therapy, cetuximab, a monoclonal antibody directed against the EGFR receptor, is currently being used in Phase III trials. Immunotherapy and vaccines may play a role in the management of oropharyngeal squamous cell carcinoma in the future but are not currently being used.

The Role of HPV and p16 in Oropharyngeal Squamous Cell Carcinoma

The Role of HPV and p16 in Oropharyngeal Squamous Cell Carcinoma.

Somewhere in the neighborhood of 85% of people will be infected by HPV during their lifetime. Many of these infections are cleared in the immunocompetent host, as we have learned in cervical disease. With respect to head and neck squamous cell carcinoma, HPV can be isolated in approximately 25% of all cases. Of all head and neck sites, HPV has the strongest etiopathogenic link to the oropharynx—most commonly HPV-16. As mentioned previously, these patients are usually younger, lack a history of significant tobacco and/or alcohol exposure, and possibly partake in "high-risk" sexual practices.

Human Papillomavirus (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. These genotypes have different profiles, including variable tissue trophism and biologic roles in human disease. The different genotypes are broken into low- and high-risk categories. The more common low-risk genotypes are 6 and 11 while the more common high-risk genotypes are 16, 18, 31, and 33. Low-risk genotypes are frequently associated with benign neoplasms such as squamous papillomas; on the other hand, high-risk genotypes are more commonly associated with malignant neoplasms such as squamous cell carcinoma.

HPV in OPSCC Oncogenesis — Genome

The HPV circular genome is usually around 8,000 base pairs in length and contains approximately 80 genes, which are categorized as early-designated as “E” or late – designated as “L”. The designations of early and late refer to the temporal sequence of gene expression. Upon entrance into the cell nucleus, HPV early genes, E1, E2, E6, and E7, are pivotal in establishing approximately 20-100 intranuclear viral DNA episomes. As the basal cell undergoes division, viral episomal replication will simultaneously occur. Whereas infected cells will continue to undergo division even as they make their way into the suprabasal layers, uninfected cells will not divide and exhibit maturation that we observe in the strata of benign squamous epithelium. As infected cells migrate towards the surface, there is upregulation of early and late genes; ultimately, fully formed virions including capsid proteins are ready for release.

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. In fact, it appears that E6 and E7 must be derived from high-risk genotypes to actually induce and transform cells in vitro.

Late genes, L1 and L2, are capsid proteins that appear to require initiation by the promoter p670.

HPV in OPSCC Oncogenesis — Pathways

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. This is assumed to play a role in the evasion of cell death, a form of cellular immortalization if you will. E6 is also thought to work outside of the p53 pathway by activating telomerases.

The E7 protein, on the other hand, binds and inactivates proteins of the retinoblastoma gene family of tumor suppressors. This family includes retinoblastoma, p130, and p107. In the normal state, retinoblastoma protein is hypophosphorylated and then binds and becomes a regulator of E2F, a transcription factor that modulates activity of other factors involved in cell cycle progression. E7 essentially binds and inactivates the hypophosphorylated retinoblastoma protein and permits E2F to proceed with cell cycle progression. Retinoblastoma will also modify the activity of other tumor suppressor proteins such as p16, a cyclin-dependent kinase inhibitor.

p16, the cell cycle regulatory protein, is up-regulated by HPV oncoproteins. To this end, as you well know, p16 is widely considered a surrogate marker for HPV infection.

HPV in OPSCC Oncogenesis — Detection

Depending on the study, HPV can be detected in 50-80% of cases of oropharyngeal squamous cell carcinoma. There appears to be at least a gradual increase in HPV detection over recent years; this is likely related to the fact that recent studies are more likely to have employed multiple testing platforms for HPV detection (ie in situ hybridization, polymerase chain reaction, reverse-transcriptase polymerase chain reaction, and so forth).

This is a good time to mention that the mechanism of disease in HPV-negative tumors diverges from what we see in HPV-positive tumors. HPV-negative tumors have shown elevated expression of cyclin D and EGFR, proteins that are often associated with tumorigenesis. Moreover, chromosomal aberrations, microsatellite instability, and epigenetic changes are more commonly seen in HPV-negative cases. These HPV-negative cases are thought to be more laden with genetic mutations induced by carcinogen exposure (such as cigarettes, alcohol, betel nut). These carcinogen-associated cases show a p53 mutation rate of more than 50%, whereas p53 mutation in HPV-positive of oropharyngeal squamous cell carcinoma, on the other hand, is rare.

Indications for HPV and p16 Testing in Oropharyngeal Squamous Cell Carcinoma

Indications for HPV and p16 Testing in Oropharyngeal Squamous Cell Carcinoma.

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. This data typically refers to patients when treated with concurrent chemoradiotherapy. It has been hypothesized that the improvement in survival is related to escalated sensitivity to radiotherapy. However, some studies suggest that the survival benefit may be observed even in the absence of radiotherapy, with surgical intervention alone. In other words, the improved prognosis may be independent of treatment modality. Nevertheless, there is compelling evidence to suggest that HPV-associated oropharyngeal squamous cell carcinoma fares better than cases that are HPV-negative.

HPV testing may also be employed in the setting of metastatic squamous cell carcinoma of unknown origin. The classic scenario is metastatic squamous cell carcinoma to a cervical lymph node. These specimens are often labeled, "neck mass."

Prognostic

HPV detection using HPV in situ hybridization is considered to be the gold standard by many clinicians—but certainly not all of them. We will address the advantages and disadvantages of HPV in situ hybridization shortly. Using in situ hybridization, HPV DNA can be seen as episomal, fully integrated, or both. The episomal pattern is characterized by diffuse nuclear staining, representing an accumulation of episomal sequences. This is precisely what we see in this micrograph; sheets of neoplastic cells with diffuse nuclear staining, almost smudgy in appearance. Naturally, the assay in this micrograph is directed against high-risk HPV genotypes. In our experience at Mayo Clinic, this is the most common pattern of HPV-positivity in oropharyngeal squamous cell carcinoma. One can often accurately interpret a case as positive even before placing the slide on the microscope stage, simply recognizing the diffuse blue or purple haze in areas of malignant tumor.

As mentioned previously, HPV DNA in situ hybridization can also reveal a fully integrated pattern. This is recognizable by punctate nuclear signals as you can appreciate in this micrograph. Although several studies have used HPV in situ hybridization as a means of classifying tumors as HPV-positive or negative, the pattern of positivity is rarely addressed. That is, little effort has been made to distinguish episomal from fully integrated from fully mixed patterns of positivity. It has been shown in squamous cell carcinoma cell lines that E6 and E7 can be expressed in the setting of both episomal and integrated HPV DNA. Currently, it is unclear if these patterns fall out into different prognostic or therapeutic categories.

p16 is a cyclin-dependent kinase inhibitor. When the HPV E7 oncoprotein inactivates retinoblastoma, p16 expression increases dramatically. It is worth noting that p16 overexpression is not exclusive to this mechanism and can be seen in 10-20% of cases of oropharyngeal squamous cell carcinoma in the absence of confirmed HPV infection. In other words, p16 expression is not a perfect surrogate marker for HPV infection because it lacks a certain degree of specificity.

However, recent studies have suggested that p16 expression is an independent prognostic factor of improved locoregional control and disease free survival in oropharyngeal squamous cell carcinoma. These studies point to the finding that HPV-negative cases that are strongly and diffusely immunoreactive for p16 show better outcome than p16 negative patients, regardless of their HPV status. This remains an area of interest as knowing the answer to this question is paramount to future testing practices in oropharyngeal squamous cell carcinoma. If p16 positive/HPV positive patients perform differently than p16 positive/HPV negative patients, it will be worthwhile to test both p16 and HPV. On the other hand, if the distinction in outcome can be forecasted by p16 immunohistochemistry alone then HPV testing is essentially futile.

In addition to serving as strong prognostic tools, HPV in situ hybridization and p16 immunohistochemistry can also provide tremendous utility in determining the primary source of metastatic disease. 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. Of note, it is important to recognize that malignant transformation of a branchial cleft cyst is exceedingly rare; in fact, some head and neck pathologists question whether such an event transpires at all.

In sum, metastatic squamous cell carcinoma to the neck that exhibits HPV and p16 positivity should be regarded as a metastasis from the tonsil or base of tongue until proven otherwise. Naturally, making the “diagnosis” of metastatic oropharyngeal squamous cell carcinoma can save precious time and resources in this cohort of patients.

Interpretation of HPV and p16 Testing in Oropharyngeal Squamous Cell Carcinoma

Interpretation of HPV and p16 Testing in Oropharyngeal Squamous Cell Carcinoma.

Histology is an insufficient predictor of HPV infection. The tumors are classically nonkeratinizing and show well-defined nests or sheets, often with comedo necrosis, that are composed of densely packed tumor cells with little intervening stroma. The cells are basaloid with hyperchromatic nuclei, high nuclear-to-cytoplasmic ratios, and ill-defined cell borders. Mitotic activity is often brisk and cells undergoing apoptosis is not infrequent. These findings are suggestive of HPV infection, but certainly not definitive.

Several HPV detection methods exist, each of them carrying a different sensitivity and specificity profile. If we do not take this into account when we engage in conversations with our clinical colleagues we are liable to create both confusion and frustration. I would argue that understanding the means of HPV detection is more valuable than the detection of HPV itself.

Interpret the Presence or Absence of HPV

This is where you want to be. First, interpret the presence or absence of HPV while giving consideration to the advantages and disadvantages of the detection method used. Second, formulate an intelligible opinion on the significance of your results.

Don't Jump the Gun

This is where you do not want to be. Don't jump the gun. Don't read a surgical pathology report or surgical pathology journal and simply look for the designation, "HPV-positive" or "HPV-negative."

Polymerase Chain Reaction (PCR)

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.

Depending on what you are referring to, the specificity of PCR can be regarded as high or low. This assay is specific in that the primers may be designed for a unique sequence of interest (ie primers that recognize consensus sequences or even sequences of particular genotypes). On the other hand, the specificity may also be regarded as low because its sensitivity is so great that viral DNA from histologically normal mucosa or contaminants can potentially give rise to false-positive results.

Unfortunately, PCR results cannot be correlated with light microscopic evaluation. Results are essentially regarded as positive or negative, there is no way of determining if the detected transcripts are from diseased or normal tissue, or even a contaminant. In this same vein, HPV detection by PCR gives no information about episomal or integrated status. Furthermore, it grants no insight into the expression status of the viral genome.

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

DNA In Situ Hybridization

DNA in situ hybridization for HPV is often considered the gold standard for HPV detection. The biggest knock on HPV in situ hybridization is its low sensitivity; there are a sufficient number of false-negatives that should inspire you 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 (ie, low- or high-risk genotypes). Aside from artifactual staining, the presence of episomal or integrated staining patterns can be considered positive with confidence. Because this assay is interpreted using the light microscope, it not only allows for the detection of viral DNA, it provides insight into the localization of HPV DNA as well. That is, in situ hybridization permits the determination of episomal or fully integrated localization of HPV DNA. In situ hybridization is readily available in most laboratories and a fair number of laboratories belonging to institutions with smaller case volumes. Importantly, in situ hybridization gives no indication of biologic activity. That is, the presence of HPV DNA tells you little if anything, about its role in the initiation and/or maintenance of malignant transformation.

E6/E7 Messenger Ribonucleic Acid (mRNA)

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.

Relatively new systems allow for examination with the light microscope which grants obvious advantages.

Unfortunately, this can be a cumbersome test to perform and is not available in the vast majority of laboratories on a clinical basis.

P16 Immunohistochemistry

Most studies have found a strong correlation between p16 immunoreactivity and HPV positivity by PCR or ISH. In other words, p16 has a high level of sensitivity.

On the other hand, p16 can be overexpressed even in the absence of HPV DNA or E6/E7 transcripts. For this reason the specificity remains a weakness.

Naturally, p16 interpretation is by way of light microscopy so the histologic correlation goes without saying.

Lastly, not all but a fair number of immunohistochemistry laboratories offer p16 immunostaining.

HPV and p16 Testing in Oropharyngeal Squamous Cell Carcinoma in Review

HPV and p16 Testing in Oropharyngeal Squamous Cell Carcinoma in Review.

Review

We reviewed the anatomy of the oropharynx; if you refuse to remember the anatomic borders of the oropharynx, simply remember that it includes the base of tongue and bilateral palatine tonsils.

The distribution of oropharyngeal squamous cell carcinoma cases is no longer exclusive to those with long-term carcinogen exposure. It now includes the so-called HPV-associated cases of oropharyngeal squamous cell carcinoma. These patients are typically younger, lack a history of significant tobacco and/or alcohol exposure, and have engaged in “high-risk” sexual behavior.

The incidence of oropharyngeal squamous cell carcinoma is rising even though cigarette use is declining. Again, this is thought to primarily be a function of the HPV-associated cases of oropharyngeal squamous cell carcinoma.

HPV is an encapsulated, nonenveloped, double-stranded DNA virus, with over 100 known genotypes. These genotypes are broken into low- and high-risk categories. High-risk genotypes, especially HPV-16, are commonly associated with oropharyngeal squamous cell carcinoma. HPV can be detected in 50-80% of cases of oropharyngeal squamous cell carcinoma.

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. It appears that E6 and E7 must be derived from high-risk genotypes to actually induce and transform cells in vitro.

What questions should you ask yourself when you evaluate HPV testing and what lies ahead for the management of oropharyngeal squamous cell carcinoma?

The presence of HPV DNA tells us nothing about transcription. Transcription tells us nothing about posttranscriptional regulation. To this end, what have the aforementioned assays told us?

Is p16 a better predictive biomarker than HPV? This has yet to be clarified definitively.

Will HPV or p16 status be incorporated into current staging practices? Deescalating treatment intensity may be an option given the less aggressive behavior of HPV-associated oropharyngeal squamous cell carcinoma.

Questions regarding the HPV vaccine, Gardasil, as a means of preventing head and neck squamous cell carcinoma are beginning to surface. Naturally, this makes the need to clarify HPV’s role in oropharyngeal squamous cell carcinoma imperative.


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