AUTHOR: John R. Basile DDS, DMSc
AUDIENCE: Dentists and dental hygienists
ABSTRACT: Recent epidemiological evidence has pointed to an alarming increase in the number of oropharyngeal squamous cell carcinomas (OPSCC). This increase is found in a population slightly younger than what has been seen for more traditional oral squamous cell carcinoma (OSCC) of the lateral tongue, base of the tongue, and floor of the mouth, which has actually seen a decline in incidence. Because infection with high-risk human papillomavirus (HPV) is known to cause genital cancers, this virus has been investigated as a possible cause of OPSCC. This course presents evidence from the scientific literature regarding the link between infection with this epithelial-tropic virus and this subtype of cancer. The mechanistic events occurring within a cell that lead to transformation to cancer will be discussed, along with the clinical and histopathological appearance of HPV-induced OPSCC, techniques for diagnosis, and patient prognosis. Issues regarding epidemiology will be presented, along with what patients need to know regarding this increasingly significant public health issue.
To understand the following from an oral healthcare perspective:
- That some viruses have the potential to cause cancer.
- How infection with a subset of HPV can transform ordinary epithelial cells into cancer.
- The clinical and histopathological characteristics of HPV-induced cancers, and the tests used to prove viral infection.
- The epidemiology of HPV-induced cancers, and why they may be increasing.
- The public health relevance of HPV infection.
CLINICAL CATEGORY: Oral-Systemic
CE ACTIVITY: Online/Self-Instructional
TOTAL CREDITS: 2 CE Credits
TOTAL COST: $20.00
PUBLISH DATE: September 10, 2013
EXPIRATION DATE: September 10, 2016
The term ‘transformation’ in cell biology refers to the change of a normal, genetically stable cell to one that is malignant or cancerous in nature. This generally occurs through environmentally induced damage to cell DNA, like when carcinogens from cigarette smoke induce mutations in the DNA of epithelial cells found inside the oral cavity. This type of change in cell biology is what leads to oral squamous cell carcinoma (OSCC); however, the same result can be achieved through infection from certain viruses—not through DNA damage—but through expression of viral gene products that usurp the ability of the cells to protect themselves from dysregulated growth and cell division. For this reason, cancer is also referred to as ‘neoplasia,’ or ‘new growth’.
HISTORY: VIRUS-CAUSING CANCERS
The concept that viruses can cause cancer is not new. The first evidence of such a phenomenon was discovered by Dr. Francis Peyton Rous of the Rockefeller University in 1910 with the publication of his work, “A transmissible avian neoplasm (sarcoma of the common fowl)”. He followed this up with work suggesting that the cancers he observed in chickens were not a result of transplantation by tumor cells, but instead were caused by an infectious agent. What he had discovered later came to be called the Rous sarcoma virus, which over-expresses a gene product called src that transforms cells of the connective tissue into cancer. Mutations in src are now associated with several human cancers as well . Moreover, by discovering the concept of viral-induced carcinogenesis, Dr. Rous received the Nobel Prize in Medicine in 1966.
Unlike DNA damaging agents, most viruses transform cells through similar mechanisms; specifically, they code for proteins that inactivate endogenous cellular tumor suppressors (which are genes that restrict cell proliferation), and/or express oncogenes that promote autonomous cell growth in the absence of normal mitogenic signaling.
Some viruses associated with human cancers include:
Human T-lymphotropic virus (HTLV)—associated with adult T cell leukemia
Kaposi’s sarcoma associated herpesvirus (KSHV)—also known as human herpesvirus-8 (HHV-8)—associated with Kaposi’s sarcoma, multicentric Castleman’s disease, and primary effusion lymphoma
Merkel cell polyomavirus (MCPyV)—found in Merkel cell carcinoma, a particularly aggressive skin cancer
Epstein–Barr virus (EBV)—linked to Burkitt’s lymphoma, Hodgkin’s lymphoma, post-transplantation lymphoproliferative disease and nasopharyngeal carcinoma.
It should be noted that many of these viruses are found in healthy adults despite not having any signs of cancer. Whether or not infection from a virus with oncogenic potential will actually lead to cancer is difficult if not impossible to predict, as it depends upon a number of different factors, including: geographic location, viral and host genetics, immunocompetency of the infected individual, and the duration and intensity of exposure to the infectious agent.
HUMAN PAPILLOMAVIRUS (HPV)
Human papillomaviruses (HPV) are a family of small viruses with a pronounced tropism for epithelial cells. More than 120 HPV have been identified to date, and most code for between 8 and 10 proteins in their double-stranded circular DNA genome. HPVs have been sorted into genus alpha, which exhibits a mucosal tropism, and genus beta that infect cutaneous cells. Infection of mucosa with ‘low-risk’ HPV—commonly type 6 and 11—usually manifests as papillomatous hyperplasia, or the common wart.
A subset of these viruses, however, referred to as ‘high-risk’ (most commonly 16, 18 and 31 in the United States and Europe) were eventually linked to cancer by Dr. Harald zur Hausen—now a professor emeritus at the University of Heidelberg—who took note of the fact that genital warts (caused by the sexually transmitted HPV infection), had a similar epidemiological pattern to that of cervical cancer. This was no small discovery, as it suggested cervical cancer is essentially a sexually transmitted disease that could be prevented with changes in sexual behavior. Although this finding was at first highly controversial, it led to Dr. zur Hausen receiving the 2008 Nobel Prize in Medicine, and spurred the development of an HPV vaccine.
MECHANISMS OF TRANSFORMATION
If cancer is caused by dysregulated or improper cellular proliferation, then many cancers can be characterized by the replicative mechanisms that govern when a cell divides (a process known as cell cycle control) which are disrupted. As stated earlier, whether or not a cell will divide is determined by the proteins coded from the tumor suppressor genes and oncogenes within that particular cell. The function and levels of these proteins are based upon—among other things—signals received from the surrounding environment that tells the cell when it is appropriate to divide or not, or when to stop dividing if it already has begun to do so.
For example, cardiac myocytes (once formed in the heart) will never receive the proper combination of signals that would alter their genetic program to induce cell division (even in the case of a heart attack), whereas epithelial cells will commonly receive these signals. In fact, with the epithelial cells of the skin at the site of injury, growth balance is tipped towards cell division until told otherwise. It is in this context that much has been learned regarding the nature of tobacco- and HPV-related oral carcinogenesis.
The primary regulators of the cell cycle are:
the tumor suppressor proteins retinoblastoma (Rb), p53, p21, p16;
the cyclins; the cyclin-dependent kinases (CDKs);
the transcription factor E2F.
These form a type of network or circuit, where one protein stimulates or inhibits another to determine the ultimate fate of the cell. As shown in Figure 1 (below), E2F will stimulate a cell to replicate its DNA and commit to cell division unless inhibited by the tumor suppressor Rb. The importance of this relationship is demonstrated by the terrible tumor from which the Rb protein derives its name—Retinoblastoma. This refers to malignancy of the retina which is usually diagnosed in children, and arises as a result of mutation and/or loss of function of Rb, leading to unrestricted cell division driven by E2F. The ability of Rb to inhibit E2F is controlled by the cyclin/CDK pairs. CDKs are ‘kinases,’ or proteins that place phosphorous groups onto Rb, which also inhibits it. The cyclins are necessary for the proper function of the CDKs, and as their name implies, they periodically accumulate or become degraded depending upon where in the cycle the cell resides.
FIGURE 1: Cell cycle control and the mechanisms of non-HPV and HPV-mediated cellular transformation.
The image above depicts a simplified model of the key players that are involved in cell cycle control. E2F activates a series of genes that promote cell division. Its activity is restricted by the tumor suppressor Rb which, in turn, is deactivated by the cyclin/CDK proteins, thereby having the effect of promoting DNA replication and cell division. The cyclin/CDKs are inhibited by the tumor suppressors p16 and p21 (among others), proteins which are upregulated by p53, the ‘guardian of the genome.’
In tobacco-induced OSCC, carcinogens induce mutations in the genes coding for p53 and p16, inactivating them and leading to loss of inhibition of the cyclin/CDKs, which results in cell division. In HPV infection, the virus promotes cell division, and by default replication of its own DNA, by inactivating p53, p21 and Rb through the activity of the early viral oncoproteins E6 and E7. It is deregulated production of E6 and E7, occurring when the virus genome integrates with that of the host, that leads to uncontrolled cell growth and cancer.
When cyclins have accumulated, the CDKs will function by phosphorylating and inhibiting Rb, which frees up E2F to promote DNA replication (Figure 1); however, the cyclin/CDKs are, in turn, inhibited by a series of tumor suppressors, which include p16 and p21. When these inhibitors are at high levels in the cell, the cyclin/CDKs are held in check and the cell does not divide. In addition, levels of p16 and p21 are controlled by p53, also known as ‘the guardian of the genome’ (Fig. 1); moreover, p53 is important because it stimulates the production of many tumor suppressors, which restrict growth and even trigger cellular suicide—a process called apoptosis. This occurs when enough genetic damage has accumulated in the cell and the chance for transformation to cancer is more likely. Mutation and loss of p53 function is one of the most frequently observed somatic genetic events in all human cancer, and when patients report that “cancer seems to run in our family,” there is often an inheritable deficiency of p53 or one of its tumor suppressor targets that is responsible .
In tobacco or non-HPV related OSCC, the cause is often direct DNA damage and mutation of a tumor suppressor (such as p53), and particularly p16 from the harmful chemicals in cigarette smoke.[5-6] Following the pathway in Figure 1, it is clear that the loss of either would lead to a loss of suppression of the cyclin/CDKs—which would phosphorylate and inhibit Rb, allowing E2F to turn on the genes necessary for uncontrolled progression through the cell cycle.
In infections with high-risk HPV, the mechanism is different, but the result is still similar. Instead of mutational inactivation of tumor suppressors, HPV inactivates the cell’s tumor suppressor proteins by making the “early” (E) gene products, which are known as the E6 and E7 oncoproteins. E6 and E7 induce normally growth-arrested, differentiating parabasal keratinocytes (the cells of the skin or mucosa that are located directly above the proliferating basal cell layer and normally do not divide), but in this case will continue to divide by inhibiting p53, p21 and Rb, respectively (Figure 1). From there, the virus is then able to usurp the cell’s DNA replicative machinery in order to copy its own DNA. This makes evolutionary sense for a virus being that it is an obligate intracellular parasite with no machinery of its own to synthesize DNA.
Once many copies of the viral genome have been made, the “late” (L) genes take over to coordinate virus particle production. A mature virus is then assembled and sheds itself from the host in sloughed epithelial cells for further continuation of the viral life cycle. At that point, the HPV genome is maintained and replicated as an independent circle of DNA (or an ‘episome’ in the host cell), and is not incorporated into the genome of the host cell. However, if a terminal event occurs in the viral life cycle, the genome rarely integrates into the host DNA, which results in the disruption of the gene coding for E2—an early gene that controls E6 and E7 expression. Uncontrolled co-expression of E6 and E7 leads to enhanced degradation of the tumor suppressor proteins p53, p21, and pRB. There are also significant differences between E6 and E7 at the nucleotide and protein level among low-risk and high-risk viral subtypes, which allow some HPVs to promote the development of cancer. In fact, it has been shown that overexpression of high-risk E6 and E7 proteins is all that is needed in order to transform an epithelial cell into cancer.
CLINICAL APPEARANCE, HISTOPATHOLOGY & DIAGNOSIS
Conventional tobacco OSCCs are usually found in gravity-dependent, poorly keratinized cells found on the lateral and ventral tongue and floor of the mouth. This is where carcinogens can accumulate and cells become particularly vulnerable to damage. Clinically, advanced OSCC appears in these areas as a large, non-healing, painless ulcer with raised, rolled borders. Histopathologically, they exhibit great variation, but to one degree or another, they demonstrate sheets of large polygonal squamous cells with distinct cell borders, and keratin formation that invades underlying connective tissues (Figure 2A).
FIGURE 2A & 2B: Keratinizing and Non-Keratinizing OSCC: A) An example of a keratinizing OSCC—such as the type usually found in smoking induced cancer—exhibits islands of broad, polygonal, squamous epithelial cells that invade underlying connective tissue. In some areas, these cells make keratin, or the ‘swirling,’ pink areas within the epithelium (better seen on the inset). B) An example of a baslaoid OSCC is more common in HPV-positive cancers. In contrast to the lesion in ‘A’, this lesion exhibits more mitotic activity and narrower, poorly differentiated blue-ish cells with indistinct cell borders and less cytoplasm. This lesion also demonstrates comedonecrosis, or cell death at the center of invading islands (more detail on the inset). Original magnification for both images is 200X; inset is 400X.
This is in contrast to HPV-induced OPSCC which are more difficult to visualize in the posterior oral cavity, such as the tonsils, the base of tongue, and the soft palate. When biopsied, HPV-induced carcinomas are often composed of smaller, non-keratinizing cells with features of comedonecrosis, or cell death in the center islands of rapidly dividing, blue ‘basaloid’ cells (Figure 2B).[8-10] Because it is a virally-induced lesion, koilocytes can sometimes be seen–demonstrated by squamous epithelial cells that have a perinuclear clearing and wrinkled, dense nuclei; as a result, it produces a viral cytopathic effect. It should be noted that basaloid features in a tumor and the presence of cells resembling koilocytes are subjective characteristics of a biopsy, and that they can also be seen in non-HPV cancers; therefore, they are not sensitive or specific tests for determining HPV infection.
Fortunately, there are other, more accurate methods to determine the presence of virus in tissue samples, although they all have their own advantages and disadvantages. These include electron microscopy—where viral particles can be seen within a cell of a productive HPV infection (although specific typing cannot be done) and polymerase chain reaction (PCR)—which detects the presence of viral DNA in tissue extracts from the binding of complimentary DNA strands. These are then amplified and read with an agarose gel inside the lab.
Comparing the two diagnostically, each method is unfortunately linked to significant limitations. In many ways, electron microscopy is less practical because it is difficult, time-consuming, and more expensive to conduct. PCR, on the other hand, is specific to only certain subtypes of viruses. In addition, the results are highly reliant upon proper tissue handling, and the skill of the technician who is performing the test. PCR also has the disadvantage of being too sensitive, and therefore is prone to false positives. Also, it can detect DNA at very low levels of a virus, which can produce results that might not actually have any biological significance.
A similar technique to PCR is called in situ hybridization (ISH). ISH also utilizes a complimentary strand of DNA—this time as a probe bound directly to viral DNA—which is present in formalin fixed paraffin embedded (FFPE) tumor tissues, the vast majority of biopsies. A more practical technique (and the one most often used in locating the presence of HPV in tumor biopsies), is known as immunohistochemistry (IHC) for p16. As shown in Figure 1, p16 is a tumor suppressor protein often lost in tobacco-induced cancers. However, in HPV infection, p16 is upregulated by the cell to compensate for the loss of p53, p21 and Rb.
IHC for p16 therefore uses antibodies to bind directly to protein preserved in FFPE tissues, and can be combined with the analysis of Ki-67 expression (a marker of cell division) as well as p53 for a surrogate test of infection. In an HPV-positive lesion, the IHC staining profile would show high levels of expression of p16, as well as enhanced cell division, elevated Ki-67, and a lack of p53 (from being degraded by the virus). The drawbacks of this method are that positive results are not always specific for HPV, and that some IHC results are equivocal and difficult to interpret. (A summary of the different diagnostic tests for the presence of HPV is shown in Table 1 [below]).
TABLE 1: SUMMARY OF TESTS FOR DETECTION OF HPY
Different methods used for detection of the presence of HPV in tissues is shown (PCR, polymerase chain reaction; IHC, immunohistochemistry; ISH, in situ hybridization; FFPE, formalin fixed, paraffin embedded).
Currently, there is no consensus on a ‘gold standard’ among the numerous detection methods, nor is there an agreement to what a positive test means for patient management, making treatment planning very difficult. Overall, p16 IHC seems to be the best test for active HPV infection because it has the highest correlation to the prognosis of OPSCC—more so than PCR or other highly sensitive (but less specific) techniques. Moreover, p16 IHC staining protocols and the antibodies available are also fairly standardized after being used and refined over the span of many years. Eventually, as technology improves, direct testing for E6 and E7 mRNA might be the most accurate indicator yet, since these are the functional oncoproteins of HPV, and have the most biological relevance to the disease. At this time, such a procedure remains impractical based on current technology.
ASSOCIATION WITH PROLIFERATIVE VERRUCOUS LEUKOPLAKIA
Proliferative verrucous leukoplakia (PVL) was defined by Hansen et al. as a slow-growing, persistent hyperkeratosis that tends to spread and become multifocal, which eventually progresses to carcinoma. It is a distinct clinical form of oral leukoplakia that resists all types of therapy, and therefore presents a great challenge to the clinician. For example, a typical patient is an elderly female without risk factors for OSCC, who has undergone repeated biopsies for leukoplakia over a long time span. The diagnosis has become more and more serious each time, progressing from verrucous hyperplasia to dysplasia to verrucous carcinoma and/or conventional squamous cell carcinoma. The diagnostic criteria are not well established, so many patients are diagnosed many years after the first presentation of the disease (which retrospectively is based upon past behavior of the lesion).
Because of its warty appearance, complex diagnosis, and lack of association with traditional OSCC risk factors, there have been great efforts to link PVL with a viral etiology, including EBV and HPV infection. Unfortunately, studies have been inconclusive, ranging from an 89% rate of HPV detection in one study to 0% in others.[14-16] Most of these studies have suffered from poor diagnostic criteria for PVL and small sample sizes, so more research will need to be conducted.
TRENDS AND EPIDEMIOLOGY
OSCC arises from the mucosa of the oral cavity. It is also the eighth most common cancer among men in the U.S., and the 14th most common cancer among women. In recent years, while cases of OSCC have decreased, OPSCC incidence has increased, particularly among groups without typical risk factors (such as tobacco or alcohol use), which leads some researchers to seek out other etiological agents.
The epidemiological evidence linking HPV exposure to OPSCC is strong. Similar to what is observed in genital HPV, the oral HPV infection is predominantly transmitted through sexual behavior, and the chance of detecting the virus in the oral cavity is correlated to an increasing number of lifetime sexual partners, in addition to early engagement of sexual activity. Overall, oral HPV infection is significantly higher among men, which is consistent with their higher rates of HPV-positive oral cancers. The reasons for this are not fully understood, but could be from the higher probability of HPV transmission through oral sex, or from higher seroconversion rates in women due to genital infections that confer greater protection against subsequent oral infections. Interestingly, the incidence of HPV also increases with the use of smoking, possibly as an immunosuppressive effect that causes direct damage to the barrier function of the oral mucosa, or from mutagenic effects of the tobacco.
Coincidently, the rate of HPV-related OPSCC (base of tongue, lingual tonsil, tonsil, oropharynx, and Waldeyer ring) are increasing. From 1984 to 2004, the diagnosis rate of HPV-positive OPSCC increased by 225%, while the incidence for HPV-negative cancers declined by about 50%. As this trend continues, the annual number of HPV-positive OPSCC is expected to surpass the annual number of cervical cancers by 2020. Based on Surveillance Epidemiology and End Results (SEER) data from the National Cancer Institute and SEER Residual Tissue Repositories Program, up to 25% of all OSCC are HPV-positive (>50% of OPSCC)—of which the vast majority test positive for the high-risk HPV type 16.
The mean age for all diagnosed oral cancers is decreasing, likely as a result of the increased incidence of OPSCC in the younger population. Johnson-Obaseki, et al. reported a statistically significant decrease of 3.52 years in age of diagnosis among women, and a 3.68 year decrease in age among men within a Canadian population between 1992 and 2007. When separated by site, HPV16-positive OPSCC was diagnosed at a mean age 7.5 years younger than HPV-unrelated OSCC.
HPV-associated Basaloid, Nonkeratinizing OPSCC has a higher rate of mitosis, and is more frequently present with lymph node metastases compared to non-HPV-associated cancers (despite having a better prognosis).[21-22] It is not fully understood why this is, but it may be related to the fact that HPV-associated OPSCC does not exhibit complete loss of p53 function, which is characteristic of tobacco-associated cancers. Though inactivated by the viral oncoprotein E6, p53 is still present in low amounts, and unlike many cancers, it is not mutated. As noted earlier, p16 is in fact elevated in these lesions, which may also contribute to improved prognoses. Though HPV-positive patients respond well to chemoradiotherapy and induction chemotherapy, it is not recommended to change management for HPV-positive or HPV-negative oropharyngeal cancers until high-quality, randomized trials are completed that assess the efficacy of current treatment modalities.[23, 24]. A summary of the differences in molecular profile, histopathological appearance, clinical profile and prognosis for HPV positive OPSCC and HPV negative OSCC is shown in Table 2.
TABLE 2: HPV-POSITIVE OPSCC VS. HPV-NEGATIVE OSCC
Summary of the differences in molecular biology, histopathology and clinical characteristics
PUBLIC HEALTH RELEVANCE AND PATIENT CONCERNS
There is a worldwide cancer burden associated with HPV that is now linked to cervical, vaginal, vulvar, penile, and anal cancers. This burden is not surprising since HPV is the most common sexually transmitted infection, with approximately 20 million annual cases worldwide. This statistic far outnumbers other STDs, including chlamydia or gonorrhea. HPV is also responsible for over 600,000 cancers a year worldwide, which is also higher than any other cancer-associated infectious disease, including OPSCC.
At this point, it is difficult to pinpoint why the rate of HPV-positive oropharyngeal cancers are increasing. While it is possible that such cancers were always present, and technological advancements have now led to tests of greater sensitivity and better detection, there are a number of potential factors that could be attributed to this increase, including:
reactivation of latent infections (due to age or health-related immune deficiency)
differences in sexual behaviors across birth cohorts
increased persistence of infection among older individuals
It is also possible that we are entering a phase of a true HPV epidemic, and therefore should consider what can be done to slow the spread of infection. Sexual behavior and smoking are two known risk factors for oral HPV infection, but these behaviors have historically been difficult to modify.
The connection between oral HPV and OPSCC has been reported in the lay press with the development and marketing of the HPV vaccine, so patients may have questions regarding potential screening tests. As we have seen, each method for the detection of HPV has its advantages and disadvantages. In regards to determining the risk of developing cancer, a “positive HPV test” in a random sample of oral mucosa would have limited usefulness from a clinical perspective. Not everyone who is a carrier of high-risk HPV will develop cancer (similar to HPV infection of genital mucosa) and there currently is no way to detect an “HPV-positive premalignant” lesion. The only possible advantage of HPV testing in this sense would be to help locate the primary lesion when the first indication of malignancy is lymph node metastasis. At that point, tests that are positive for HPV would thereby suggest the primary lesion was in the oropharynx.
Today, there is a vaccine against the high-risk viral types known as HPV16 and 18 (at least for genital infections), so it is possible that vaccination could confer resistance to oral infections as well. Perhaps the best thing to do is demonstrate increased vigilance for OPSCC among the younger population through a better, more thorough intra-oral soft tissue exam and increased efforts in education.
Among the many human viruses that have been linked to cancer, HPV is noted for its ubiquity and the number of different sites in the body the virus can infect. HPV-associated OPSCC is a separate entity from tobacco-related OSCC, with a set of molecular changes occurring within the cell that leads to a unique histopathological and clinical appearance, as well as prognosis and demographic profile. HPV-associated cancers occur more commonly in the oropharynx and in younger patients compared to tobacco-related OSCC—meaning that the clinician needs to do a thorough intraoral exam in order to detect these lesions, and be more vigilant in educating patients about the risk of infection.
Instead of direct DNA damage, the HPV high-risk viral oncoproteins E6 and E7 promote malignant transformation by forcing the cell through dysregulated proliferation. Understanding these changes at the cellular level and identifying them more accurately may one day lead to advanced diagnostic tests for OPSCC. Perhaps it would also alter therapeutic regimens for this disease, considering that HPV-associated cancers respond better to treatment than non-HPV lesions, though further study is required. Finally, while tobacco-associated OSCC are becoming less common, HPV-induced OSCC are on the rise, concomitant with the potential for a worldwide HPV pandemic. Whether or not the HPV vaccine will guard against oral infection and the subtypes responsible for cancer, the trend of increasing infections and development of OPSCC remains to be seen.
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