Human Papillomavirus and Oral Cavity Cancer
Mark Zafereo, M.D.
September 4, 2008

J.O. is a 38-year-old Hispanic gentleman who presented to Ben Taub General Hospital with a 3-month history of right tongue pain.  He had no weight loss or other symptoms except odynophagia.  He had no past medical history, was a never smoker, had never used smokeless tobacco or marijuana, and drank approximately six beers per week on the weekends.

On physical examination, he was noted to have a 2.5 by 2 cm ulcerative lesion of the right lateral oral tongue which did not involve the floor of mouth or extend significantly to the base of tongue.  He had no trismus or palpable lymphadenopathy.  CT scan of the neck revealed a tumor of the right lateral oral tongue which did not significantly extend to the base of tongue, floor of mouth, or involve the mandible.

Biopsy revealed moderately differentiated squamous cell carcinoma, and he was inititally staged a T2 N0 M0 squamous cell carcinoma of the right oral tongue.  He underwent a right partial glossectomy and right levels I-III selective neck dissection.  Surgical margins were negative, but he did have 4 of 11 metastatic lymph nodes in level 3 of the right neck with extracapsular spread.  He was therefore restaged a T2 N2b M0 squamous cell carcinoma of the right oral tongue and underwent postoperative concurrent chemoradiotherapy and has remained without evidence of disease, now 8 months post-treatment.

So questions that arise include the etiology of an oral tongue cancer in a 38 year-old never smoker, never user of smokeless tobacco.  Second, how common is oral cavity cancer in this population? And, finally, can a better understanding of the etiology help us to better define prognosis and perhaps eventually management of oral cavity cancer in the young.

Today I will review incidence trends in oral cavity cancer, characterize the group of young patients with oral cavity cancer, look at human papillomavirus and evidence of an association between HPV and oral cavity cancer in young patients, and finally look at a review of HPV and young patients with oral cavity cancer at MD Anderson Cancer Center.

Worldwide there are 267,000 oral cavity cancers diagnosed yearly and it’s the sixth most prevalent cancer, ranking third in developing countries and eighth in developed countries.  In the US, there are estimated to be almost 50,000 incident invasive head and neck cancer diagnoses in 2008, roughly 20,000 of which will be oral cavity cancers. 

Data from the National Cancer Institute Surveillance, Epidemiology, and End Results database indicate that, since 1975, while rates of cigarette use (indicated by the diamonds) and laryngeal cancer (indicated by the black lines) are declining sharply, oral and pharyngeal cancer incidence (illustrated by the white lines) has declined less rapidly.  A more in depth look at this data reveals that over the last 2 decades, pharyngeal and tongue cancer absolute numbers (illustrated by the dark lines) have increased, while other oral cavity sites and larynx are declining.

Carvalho and others in 2005 also studied the SEER database, indicating that while incidence rates of larynx and oral cavity cancer are declining, rates of tongue and oropharyngeal cancer are remaining steady.

More specifically, this year, Chaturvedi and others used the SEER database to look at incidence rates of all oral cavity cancers stratified by age, demonstrating a decreasing incidence of oral cavity cancer in patients over 40, but a statistically significant increasing rate of oral cavity cancer in patients 30-39 years old.

In 2005 Shiboski and others observed from the SEER database that the incidence of oral tongue cancer among whites ages 20-44 increased sharply between 1973 and 2001, while the incidence of other oral cavity site cancers declined, suggesting that oral tongue cancer is the main driver of the overall increasing incidence of oral cavity cancer in young patients.

I went to the SEER website and looked at the incidence of overall oral cavity and oropharynx cancers and larynx cancers, which are decreasing in incidence among the 20-49 age group, which you can see on the left, and then looked at the specific incidence of tongue and oropharynx and tonsil cancer among the same young age group, which is increasing, as depicted on the right.

Squamous cell carcinoma of the oral cavity, and particularly the oral tongue, in young patients was recognized as a potential clinical entity after a seminal report by Dr. Byers at MD Anderson in 1975.  He identified oral tongue patients younger than 30 who were treated between 1956 and 1973, noting that the usual factors such as tobacco, alcohol, and poor oral hygiene were absent in this subset of patients. 

Since that time, numerous investigators have examined young oral cavity cancer patients, and often specifically young oral tongue cancer patients, noting an increasing incidence in the young age group, while traditional estimates have been fewer than 3% of all oral cavity squamous cell carcinomas occuring in patients less than 40.  In a Finnish series in 1996, Atula reported an escalating incidence in oral tongue patients less than 40, from 4% in the 1960s to 7% in the 1980s.  Similarly in 1998, Myers and others demonstrated an increase from 4% in the early 70s to 18% in the early 90s.

In 1999, Verschuur and others at Princess Margaret Hospital in Toronto performed a matched control study of 185 patients younger than 40 with head and neck SCC between 1958 and 1992.  In the younger age subgroup, they noted a significantly higher incidence of females and nonsmokers and a significantly lower propensity for development of second primary tumor, traditionally associated with smoking.  They also noted a trend for more oral cavity and oropharynx primary sites in the younger age subgroup, such that nonsmoking young patients presented with a tumor of the oral cavity in 69% of cases, with a female predominace in this group.

Funk et al from Iowa in 2002 examined about 60,000 oral cavity cancers in the National Cancer Data Base, noting 76% oral tongue cancers in the younger than 35 age group, compared to less than 36% in older age groups.

In 2002, Schantz at New York Eye and Ear looked at trends in the SEER database over a 25-year period from 1973 to 1997 and noted a 12% increase in oral tongue cancer in <40 year-old non-Hispanic whites, but decreases in incidence among african-americans and other races.  He also noted increases for both females and males, suggesting that this increase in incidence is not just due to an increase in smokeless tobacoo, since young women have an extremely low prevalence of smokeless tobacco.

While we can describe general patterns in young oral cavity patients, it’s important to keep in mind that there are likely several diverse patient groups which make up this population.  For instance, one subset of patients may be a subset of young patients with male predominace who began smoking and drinking heavily in their early teens and present with oral cavity cancer in their late 30’s, while another group would be patients that may possess a familial genetic deficit or state of immunodeficiency that predisposes them to development of earlier and more aggressive tumors.

And this may account for some of the differences in survival in young patients.  While earlier studies showed poorer prognosis for young patients, most recent studies comparing survival have shown either equal or favorable survival when comparing younger patients to older matched controls.

But, in summary, there is a large group of young oral cavity patients which tend to have a higher propensity to be females, non-Hispanic whites, nonsmokers with tongue cancers who have low incidences of second primary tumors.

So what are the known and purported risk factors for these patients?  Tobacco, including smokeless tobacco, and alcohol have long been known to be the most important risk factors in oral cavity cancer, but with absence of these traditional risk factors in a significant proportion of younger patients and the relatively short timespan for these behaviors to exert a detrimental effects, investigators have looked at other etiologic agents.  Regarding smokeless tobacco, it’s important to again keep in mind that many of these young patients are females, who have very low rates of smokeless tobacco use.  Betel nut chewing, which includes the betel leaf and the areca nut, pictured top right, with or without the inclusion of tobacoo, has also long been identified as a major risk factor for oral cancer in older Asian populations.  So called "betel Barbies”, pictured bottom right, work from roadside booths and move between traffic throughout Taiwan, selling betel nut to passing motorists.  Pan masala is a mixture of nuts, seeds, herbs, and spices which is very popular in India, and is often mixed with tobacco.  However, these products do not account for the rise in incidence of young oral cavity cancer patients in the United States and Europe, and a study in the British Journal of Cancer in 1989 showed that among oral cavity cancers in India under the age of 30, there was no association with chewing habits.  Marijuana has also been purported to be an etiologic agent, however a recent matched study of over 1,000 patients at UCLA showed no increase in any lung or aerodigestive tract cancer associated with marijuana use.  Other factors that have been speculated to play a role include laryngopharyngeal reflux, occupational exposures, immunodeficiency and genetic factors and viruses such as EBV and HPV.

So a likely candidate for this increasing incidence of oral cavity, and particularly oral tongue SCC, in the young would be HPV, which has been well documented in the literature with tonsillar and other oropharyngeal sites such as the BOT.   For example in Scandanavia, the prevalence of HPV 16 positive tonsillar carcinomas increased over 3-fold over the last three decades.  Similar data has been illustrated in Colorado, where prevalence of HPV in oropharyngeal carcinomas has increased over 4-fold over the last three decades.

Human papillomavirus is a 7.9 kb, nonenveloped, double-stranded, circular DNA virus.  A little background regarding the history of HPV begins with genital warts, which were known to the ancient Greeks and Romans but believed to be the result of syphilis or gonorrhea until the 20th century.  In 1907, Ciuffo established the infectious nature of warts by inoculating wart filtrates into skin and inducing papillomas.

In 1933, Dr. Richard Shope was able to isolate virus particles from tumors on captured wild rabbits and used these to inoculate domestic rabbits, which then developed similar tumors. He also noted that these tumors could progress to malignancy, and the virus is thought to be the source of the myth of the jackalope, a rabbit with the antlers of an antelope.

And in 1976, Zur Hausen assigned the first four types of HPV to cutaneous warts, and since that time over 100 types have been isolated and characterized, with distinctions mostly in the type of epithelium infected and the ability to effect cellular transformation into either benign or malignant disease.

Subtypes correlate with tissue preference, disease severity, and the ability to effect cellular transformation. Types 6 and 11 are associated with lowest malignant potential, commonly found in genital condylomas as well as laryngeal papillomatosis.  Types 31, 33, and 45 have intermediate malignant potential and are occasionally found in genital or oral malignancies.

Identified in the early 1980s, HPV 16 and 18 are the two most common high-risk human papillomaviruses that infect mucosal epithelial cells and induce cancer in the uterine cervix, anogenital tract, oral cavity, and oropharynx.  In oropharyngeal studies, approximately 90% of HPV-associated tumors are HPV type 16, while a slightly smaller percentage, approximately 75% of oral cavity cancers, are associated with HPV type 16. 

The genome consists of three regions: an upstream regulatory region, and two regions named according to the phase of infection in which they are expressed, the early, E, and late, L, regions.  Early genes are numbered 1-7 with E1 and E2 involved in replication of the viral genome.  The two genes of the late region, L1 and L2, encode the viral structural proteins and are the targeted proteins for vaccines.  E6 and E7 have been shown to be potent oncogenes, inducing malignant transformation in host cells, which accelerate proliferation of infected cells by disturbing the function of their respective tumor suppressor genes p53 and pRb, which is the retinoblastoma gene.  The top picture depicts the normal cell cycle with p53 and pRb functioning to arrest the cell cycle in response to DNA damage.  The bottom left picture depicts carcinogenesis in HPV negative tumors, with carcinogens in tobacco and alcohol causing mutations in the p53 or pRb pathways, while the picture to the bottom right depicts carcinogenisis in HPV positive tumors, where the same downstream effect is due to disruption and degradation of p53 and pRb by the E6 and E7 oncogenes.  So while HPV negative tumors are generally associated with mutated p53, HPV positive tumors generally have wild-type p53.

HPV infects basal cells of the epithelium, which are the only dividing cells of the epithelium, but not all tissue that are exposed to HPV become infected.  In an active infection, the late genes are expressed and virions are produced, but in a latent infection, some early genes are expressed but no viral production occurs and tissue remains clinically and histologically normal.  The reasons why either an active or latent infection occur are not completely understood, but one hypothesis is host immunity.

This is a graph of cancers attributable to HPV.  It’s been studied most extensively in the context of cervical cancer, in which its prevalence is virtually 100%, although HPV infection also has been closely linked to the majority of anogenital cancers.  Another graph includes mouth and throat cancer.  Gray bars show the total annual number of cases of various cancers worldwide, while fraction of cancers estimated to be induced by HPV is shown in red. Again, while almost all cervical and a large proportion of anogenital tract cancers are attributable to HPV, Park and others estimated only 12% of oropharynx and 3% of oral cavity cancers to be attributable to HPV, although other studies have estimated the attributable fraction for oropharyngeal cancers to be much higher at 18-47%.

In a study of the International Agency for Research on Cancer, looking at over five thousand cases of head and neck squamous cell carcinoma biopsies that used PCR-based methods to detect HPV from 60 studies in 26 different countries, the prevalence of HPV was 23% in the oral cavity, 35% in the oropharynx, and 24% in the larynx.

Focusing now on oral cavity cancers, one of the major pitfalls in the literature is that many studies do not adequately characterize tumor location, with many studies reporting HPV tumor prevalence rates for " oral cancers that include both oral cavity and oropharyngeal subsites."  Another limititation is that general patient profiles in terms of age, risk factors, and other such variables differ among different institutions and countries, making it difficult to compare studies.  Thirdly, since prevalence of HPV in oral cavity cancer has been shown to be much lower than that of oropharyngeal cancer, small subsets of patients who are at high risk for HPV-induced tumors may not be indentifiable in a much larger population of non-HPV induced tumors.

Methods of HPV detection differ among studies; ELISA is often thought to be the least reliable method for HPV detection as it uses antibodies to HPV in the serum, such that HPV could easily have been introduced from other body sites.  PCR has high sensitivity but does not differentiate between cellular infection and presence of extracellular HPV which may represent contaminating DNA or extracellular DNA that has not been incorporated into the cell.  The gold standard is perhaps amplyfying HPV transcription products such as RNA or proteins with PCR, as this suggests that the virus has actually been integrated into the host genome.  However, this is very complex and expensive, and thus few studies have performed these types of tests.

And finally, sensitivity for HPV detection can be lowered by degradation of the sample with paraffin-embedded samples.  So I’ll present a few studies with data on oral cavity cancer and HPV, with the caveat that there are very few well-done large studies in the literature focusing exclusively on HPV and oral cavity cancer.  More often, studies will look at quote oral cancers and include both oral cavity and oropharyngeal cancers.

Ritchie and others from Iowa published a study in 2003 in the International Journal of Cancer of 94 oral cavity and 45 oropharyngeal squamous cell carcinomas, testing all tumors with PCR for HPV DNA.  They detected HPV in 42% of oropharygneal tumors and 11% of oral cavity tumors.  Patients with HPV positive tumors were 4 times more likely to have a history of orogenital sex. Interestingly, patients with a history of orogenital sex were significantly younger, better educated, and had many more lifetime sexual partners than those without a history of oral sex.  Patients with HPV positive tumors were 3 times less likely to be smokers and had significantly better overall survival, 71% vs. 49% at 5 years.

Gillison and others from John Hopkins published a study in 2000 in the Journal of the National Cancer Institue of 84 oral cavity and 60 oropharyngeal squamous cell carcinomas, testing all tumors with PCR for HPV DNA, as well as southern blot and in situ hybridization.  They detected HPV in 57% of oropharygneal tumors and 12% of oral cavity tumors, noting that HPV positive tumors were more likely to be poor tumor grade and again had significantly better survival. 

In a multicenter international study published in the Journal of the National Cancer Institute in 2003, Herrero and others looked at approximately 1500 oral cavity patients, 250 oropharyngeal patients who had their tumors tested for HPV DNA using PCR.  Four percent of oral cavity and 18% of oropharyngeal tumors had HPV DNA.  Again, HPV positive tumors were less likely to be smokers and tobacco chewers and more likely to have greater than one lifetime sexual partner and a history of orogenital sex.

The authors concluded, as many others have, that HPV appears to play an etiologic role in many cancers of the oropharynx and a small subset of oral cavity cancers, but they do not suggest any particular subset of oral cavity patients that may be at increased risk.

And in fact, there have been no previous reports focusing specifically on the prevalence of HPV in young oral cavity cancer patients. So I participated in a study with Dr. Sturgis and others at MD Anderson, which we presented to the American Head and Neck Society in July, looking at the association between young oral cavity cancer patients at MD Anderson and HPV.

With increasing incidence of oral cavity and particularly oral tongue cancer in young patients, coupled with evidence that incidence of HPV-associated oropharyngeal tumors is rising, we hypothesized that this increasing incidence of oral cavity cancer in young patients may potentially be causally related to HPV.

We identified 256 newly diagnosed and previously untreated oral cavity cancer patients (excluding lip) between 1996 and 2005 from a prospective molecular epidemiology database.  The site was confirmed as oral cavity, as opposed to oropharynx, by a staff head and neck surgeon as well as based on institutional multidisciplinary treatment planning.  Almost 20% of patients in this cohort were less than 40, while about one-third were less than 50.  DNA was extracted from tumor samples and analyzed for both HPV 16 E6 and HPV 16 E7 using PCR. 

First, we looked at age of presentation stratified by HPV 16 status.  On the left the mean age at presentation for 31 patients both  HPV 16 E6 and E7 positive was 39, compared to a mean age of 58 for 225 patients who were not positive for both E6 and E7.  When looking at either HPV 16 E6 or E7 positivity, 53 patients who were positive for either HPV E6 or HPV E7 had a mean age of 46, compared to a mean age of 58 for 203 patients who not positive for either E6 or E7.  Both of these comparisons were highly significant. We quartiled age into patients less than 40 years of age, 40-49, 50-69, and greater than 69.  The grey represents patients who were both E6 and E7 negative, the green patients E6 and/or E7 positive, and the blue patients who were both E6 and E7 positive.  It’s important to first note that the percentage of HPV positive patients in the standard age group for oral cavity cancer, the 50-69 age group, is 13%, which is consistent with the literature.  However, the percentage of patients E6 and/or E7 positive and the percentage of patients both  E6 and E7 positive increases in a step-wise manner with decreasing age, such that less than 40 years of age have about a 40% incidence of HPV positivity.

There is therefore a clear, highly significant trend in age of presentation when comparing the E6 and E7 negative patients in grey to the E6 and/or E7 positive patients in green or the E6 and E7 positive patients in blue.  We also found it interesting that the vast majority of young patients (86%) who were HPV positive were positive for both E6 and E7.  When we dichotomized the age groups to patients less than versus greater than or equal to 50 years of age, we found a similar highly significant difference between patients both E6 and E7 negative, again in grey, versus those who were E6 and/or E7 positive in green and those who were both E6 and E7 positive in blue.  Thirty-one percent of patients less than 50 were both E6 and E7 positive, compared to 3% of patients greater than or equal to 50 who were both E6 and E7 positive.

We then stratified the 256 patients according to sex, ethnicity, smoking and alcohol status, subsite of oral cavity, and grade.  In both quartiled and dichotomized analyses, the dichotomized analysis shown here, we found that patients who were younger tended to be more commonly never smokers, never drinkers, and to have oral tongue cancer.  These findings were highly significant.  Looking at the graph to the left, 50% of patients less than 50 were never smokers, compared to approximately 25% of patients greater than 50 who were never smokers.  In the graph to the right, 80% of patients less than 50 had oral tongue cancer, compared to only 35% of patients greater than or equal to 50 with oral tongue cancer.

So, finally, we looked at how HPV 16 status varied with age of presentation stratified by sex, ethnicity, smoking status, alcohol status, subsite of oral cavity, and grade.  The graph to the left illustrates stratification according to sex.  Fifty percent of females less than 40 were either HPV E6 and/or E7 positive, while 42% of males less than were HPV E6 and/or E7 positive.   The same trend of increasing incidence of HPV positivity with decreasing age of presentation previously illustrated for the overall group of 256 patients is also evident when stratified according to sex. When compared to HPV E6 and E7 negative patients, the differences are highly significant.  The graph to the right illustrates stratification according to smoking status.  Forty-four percent of never smokers and 48% of ever smokers less than 40 years of age were positive  for HPV E6 and/or HPV E7.  These numbers again fall off dramatically as age increases.  When compared to E6 and E7 negative patients, the trend is highly significant.

And,  similarly, for every one of the subgroups including alcohol status, subsite of oral cavity, and grade, the trend for increasing prevalence of HPV with increasing age was highly significant.  The one exception to this was ethnicities other than non-Hispanic whites, which had the same overall trend but did not reach statistical significance because of a small sample size of 41 patients in this subgroup.

And the final point, getting back to the increasing incidence of oral tongue cancer described earlier, 44% of oral tongue patients under the age of 40 were positive for either HPV 16 E6 and/or E7, and, in fact, 36% between the ages of 40 and 49 were positive, compared to only 11% HPV positivity in patients greater than or equal to 50 years of age.

In conclusion, this study suggests HPV as a potential etiologic agent for the increasing incidence of oral cavity cancer, and particularly oral tongue cancer, in the young.  As with HPV and oropharyngeal cancer, this could potentially also have implications with regards to prognosis and eventually treatment and prevention of these tumors.  In addition to preventing oropharyneal cancer, oral cavity cancer prevention may represent another reason for HPV vaccination of young males.

As mentioned previously, one of the major pitfalls in defining the role of HPV in oral cavity cancer is the heterogeneity in the methods of testing for HPV in the literature, so future studies should focus on identifying biologically relevant infections, perhaps by the presence of E6 and E7 viral messenger RNA or looking at viral-cellular fusion transcripts indicating integrated HPV DNA, so that hopefully we can better define the etiology of oral cavity cancer in the young and perhaps develop novel treatment and prevention strategies that can potentially help patients such as the one in the case presentation today.

Case Presentation

JO is a 38- year-old Hispanic gentleman who presented to Ben Taub General Hospital with a 3-month history of right tongue pain.  He had no weight loss or other symptoms except odynophagia.  He had no past medical history, was a never smoker, had never used smokeless tobacco or marijuana, and drank approximately six beers per week on the weekends.

On physical examination, he was noted to have a 2.5 by 2 cm ulcerative lesion of the right lateral oral tongue which did not involve the floor of mouth or extend significantly to the base of tongue.  He had no trismus or palpable lymphadenopathy.  CT scan of the neck revealed a tumor of the right lateral oral tongue which did not significantly extend to the base of tongue, floor of mouth, or involve the mandible.

Biopsy revealed moderately differentiated squamous cell carcinoma, and he was inititally staged a T2 N0 M0 squamous cell carcinoma of the right oral tongue.  He underwent a right partial glossectomy and right levels I-III selective neck dissection.  Surgical margins were negative, but he did have 4 of 11 metastatic lymph nodes in level 3 of the right neck with extracapsular spread.  He was restaged a T2 N2b M0 squamous cell carcinoma of the right oral tongue and underwent postoperative concurrent chemoradiotherapy, receiving three cycles of cisplatin and external beam radiation to the primary site and neck.  He has remained without evidence of disease, now 8 months post-treatment. 

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