Evaluation of Human Papilloma Virus Diagnostic Testing in Oropharyngeal Squamous Cell Carcinoma: Sensitivity, Specificity, and Prognostic Discrimination

Source: Clinical Cancer Research


Purpose: Human papillomavirus-16 (HPV16) is the causative agent in a biologically distinct subset of oropharyngeal squamous cell carcinoma (OPSCC) with highly favorable prognosis. In clinical trials, HPV16 status is an essential inclusion or stratification parameter, highlighting the importance of accurate testing.

Experimental Design: Fixed and fresh-frozen tissue from 108 OPSCC cases were subject to eight possible assay/assay combinations: p16 immunohistochemistry (p16 IHC); in situ hybridization for high-risk HPV (HR HPV ISH); quantitative PCR (qPCR) for both viral E6 RNA (RNA qPCR) and DNA (DNA qPCR); and combinations of the above.

Results: HPV16-positive OPSCC presented in younger patients (mean 7.5 years younger, P = 0.003) who smoked less than HPV-negative patients (P = 0.007). The proportion of HPV16-positive cases increased from 15% to 57% (P = 0.001) between 1988 and 2009. A combination of p16 IHC/DNA qPCR showed acceptable sensitivity (97%) and specificity (94%) compared with the RNA qPCR “gold standard”, as well as being the best discriminator of favorable outcome (overall survival P = 0.002). p16 IHC/HR HPV ISH also had acceptable specificity (90%) but the substantial reduction in its sensitivity (88%) impacted upon its prognostic value (P = 0.02). p16 IHC, HR HPV ISH, or DNA qPCR was not sufficiently specific to recommend in clinical trials when used in isolation.

Conclusions: Caution must be exercised in applying HPV16 diagnostic tests because of significant disparities in accuracy and prognostic value in previously published techniques.

This news story was resourced by the Oral Cancer Foundation, and vetted for appropriateness and accuracy.

October, 2011|Oral Cancer News|

Researchers find potential new therapeutic strategy for head and neck cancer

Source: www.uab.edu
Author: Beena Thannickal

Shih-Hsin (Eddy) Yang, M.D., Ph.D., an assistant professor in the UAB Department of Radiation Oncology and associate scientist in the experimental therapeutics program at the UAB Comprehensive Cancer Center, found a way to prevent head and neck cancer cells from repairing damage to DNA as they grow.

The findings, published by the Public Library of Science, showed that using the drug cetuximab can induce a DNA repair defect in head and neck cancer cells, and subsequently render the tumors susceptible to PARP inhibitors, which block enzymes that repair some types of DNA damage. This method prevents cancer cells from repairing the damage to the DNA as they grow, ultimately leading to cancer inhibition.

Poly ADP-ribose polymerases, or PARPs, are enzymes that repair some types of damage done to DNA. If they are inhibited, a backup repair pathway is initiated. Cetuximab, which inhibits the epidermal growth factor receptor signaling pathway of cancer cells, blocks this backup pathway and thus induces cancer cell death.

“The novelty of this finding is that we use targeted agents like cetuximab, in combination with a PARP inhibitor, ABT-888, both of which have already been tested to be safe in humans, to selectively kill tumors defective in DNA repair while potentially minimizing side effects,” says Yang.

Cetuximab was pioneered by James Bonner, M.D., chair of the UAB Department of Radiation Oncology, in a landmark multi-institutional clinical trial in head and neck cancer patients.

Because head and neck cancers are frequently aggressive, outcomes for patients are currently poor.

“This new potential therapeutic strategy may improve outcomes while keeping a favorable side effect profile,” says Yang.

September, 2011|Oral Cancer News|

A step toward a saliva test for cancer

Source: www.sciencedaily.com
Author: staff

A new saliva test can measure the amount of potential carcinogens stuck to a person’s DNA — interfering with the action of genes involved in health and disease — and could lead to a commercial test to help determine risks for cancer and other diseases, scientists reported in Denver during the 242nd National Meeting & Exposition of the American Chemical Society (ACS).

“The test measures the amount of damaged DNA in a person’s body,” said Professor Hauh-Jyun Candy Chen, Ph.D., who led the research team. “This is very important because such damaged DNA — we call this ‘DNA adducts’ — is a biomarker that may help doctors diagnose diseases, monitor how effective a treatment is and also recommend things high-risk patients can do to reduce the chances of actually getting a disease,” said Chen. The research team is at National Chung Cheng University (NCCU) in Taiwan. “We tried urine and blood and found these adducts. Then we turned our attention to saliva. It’s much more convenient to collect a sample of saliva.”
A DNA adduct forms when a potentially cancer-causing substance is chemically attached to a strand of DNA, which makes up genes. People come into contact with such substances in the environment, certain workplaces and through everyday activities. Cigarette smoke, for instance, contains at least 20 known cancer-causing substances. When such a substance binds to DNA, it changes the DNA so that genes may not work normally. Our body has a built-in repair system that can naturally clear up such damage. If that system fails, however, a DNA adduct could lead to mutations or genetic changes that, in turn, could lead to cancer. DNA adducts also accumulate with aging and have been linked to other health problems, including inflammatory diseases and chronic brain disorders like Alzheimer’s disease.

The new test measures the levels of five key DNA adducts, including some that form as a result of cigarette smoking. Traditionally, DNA for such tests had to be obtained by taking a blood sample and processing the white blood cells, which contain large amounts of the genetic material. More recently, however, scientists found that DNA samples could be obtained more conveniently from saliva. The DNA is present in white blood cells found naturally in saliva and from cells shed from the lining of the mouth. Chen uses a very sensitive laboratory instrument called a mass spectrometer to analyze for DNA adducts.

Chen envisions several uses for any potential commercial version of the test, which she said would probably cost several hundred dollars. One, for example, might be health promotion among people exposed to carcinogens due to lifestyle, occupation or other factors. Detection of high levels of DNA adducts in cigarette smokers, for instance, could encourage them to stop. Follow-up tests showing a decline in DNA adducts could reinforce their healthier lifestyle.

September, 2011|Oral Cancer News|

Resveratrol Selectively Induces DNA Damage, Independent of Smad4 Expression, in Its Efficacy against Human Head & Neck Squamous Cell Carcinoma

Source: Clinical Cancer Research
Author: Robert A. Sclafani, University of Colorado School of Medicine, Campus Box 8101, Room 9100, Aurora, CO 80045. Phone: 303-724-3271; Fax: 303-724-3215; E-mail:Robert.Sclafani@ucdenver.edu



Purpose: Alterations in Smad4 signaling and its loss cause genomic instability and head and neck squamous cell carcinoma (HNSCC), suggesting that agents that target both Smad4-dependent and -independent pathways could control HNSCC.

Experimental Design: Resveratrol efficacy was evaluated against the HNSCC cells FaDu, Cal27, Det562, and Cal27-Smad4 for viability, DNA damage, cell-cycle progression, and apoptosis, as well as γ-H2AX expression, and focus formation (γ-H2AX and Brca1). Resveratrol efficacy was also examined in nude mice for FaDu xenograft growth. Xenografts were analyzed for γ-H2AX and cleaved caspase-3.

Results: Resveratrol (5–50 μmol/L) suppressed viability and induced DNA damage in FaDu and Cal27 cells but not in normal human epidermal keratinocytes and human foreskin fibroblasts, showing its selectivity toward HNSCC cells; however, Det562 cells were resistant to resveratrol even at 100 μmol/L. Cal27 cells stably transfected withSmad4 showed similar resveratrol effects as parental Cal27, indicating that a lack of resveratrol effect in Det562 cells was independent of Smad4 status in these cells. Furthermore, resveratrol caused S-phase arrest and apoptotic death of FaDu and Cal27 cells together with induction of Brca1 and γ-H2AX foci. Resveratrol (50 mg/kg body weight) treatment also inhibited FaDu tumor growth in nude mice, and γ-H2AX and cleaved caspase-3 were strongly increased in xenografts from resveratrol-treated mice compared with controls.

Conclusion: Our findings for the first time showed antiproliferative, DNA damaging, and apoptotic effects of resveratrol in HNSCC cells independent of Smad4 status, both in vitro and in vivo, suggesting that more studies are needed to establish its potential usefulness against HNSCC. Clin Cancer Res; 17(16); 5402–11. ©2011 AACR.

This article is featured in Highlights of This Issue, p. 5215

  • Received April 21, 2011.
  • Revision received June 10, 2011.
  • Accepted June 16, 2011.
August, 2011|Oral Cancer News|

Cancer vaccines make progress in combating disease

Source: http://www.masshightech.com/
Author: Lori Valigra, Mass High Tech correspondent

Sen. Edward Kennedy’s death two years ago from the deadly form of brain cancer, glioblastoma multiforme (GBM), refocused attention on how slowly treatments have progressed since former President Richard Nixon declared the war on cancer in 1971.

But a new form of treatment that goes beyond oncology drugs and surgery is now coming of age: cancer vaccines. At the recent American Society of Clinical Oncology (ASCO) meeting in Chicago — a major forum for cancer researchers — several companies and research groups reported progress on cancer vaccines, including a New England company with a shot for GBM. Agenus Inc. of Lexington reported its Prophage G-200 almost doubled the longevity of patients with recurring GBM to 11 months. That’s good news to patients with that particularly aggressive form of cancer, which also killed composer George Gershwin and music synthesizer legend Robert Moog.

Vaccines represent a relatively new approach to fighting the spread of cancer, having appeared in the last decade. The basic concept is similar to a vaccine for a disease like measles: an injection in the arm induces an immune response that helps the body fight a particular pathogen, in this case, a cancer. An effective immune response would then shrink tumors and extend lives.

Research and Markets estimates that the relatively new market for cancer vaccines could rise sharply to top $7 billion by 2015. The research company looked at six main categories of cancer vaccines: antigen/adjuvant, DNA, vector-based, tumor cell, dendritic cell, and anti-idiotype.

“Cancer vaccines are some of the most exciting areas of medicine we’ve seen in a while. They have the potential to change the way we treat cancer worldwide,” said Anthony Vasconcellos, president and CEO of InCytu Inc., a Lincoln, R.I., company (see page 8) with a very early stage vaccine that boosts the activity of dendritic cells, which in turn activate the immune system, to target GBM and other cancers. “Instead of circumventing the body’s mechanisms, the vaccines harness them. Cancer vaccines really will change the face of medicine.”

Early cancer vaccines were not potent enough, noted Thomas Davis, MD, senior vice president and chief medical officer at Celldex Therapeutics Inc. of Needham (see page 8), a cancer vaccine spinout of Medarex Inc. of New Jersey (now owned by Bristol-Myers Squibb Co.). Davis said a critical turning point for cancer vaccines was the discovery by Drs. Ralph Steinman and Zanvil A. Cohn at Rockefeller University in New York of dendritic cells, which are pivotal to the immune system. “The cells don’t work well in patients with cancer. There’s still a lot we do not know, but we are getting a better sense of how dendritic cells work and how cancer suppresses them,” Davis said. “We are in the dawning of the vaccine era.”

Garo Armen, CEO of Agenus Inc. (formerly Antigenics), agreed. In the last 12 months, especially, there has been a lot of positive activity with cancer vaccines, he said, pointing to Dendreon Corp.’s Provenge for metastatic castrate resistant (hormone refractory) prostate cancer, which was the first cancer vaccine approved by the U.S. Food and Drug Administration, and Bristol-Myers Squibb’s Yervoy for metastatic melanoma.

“These two vaccines were very important approvals in the last year,” Armen added. “To date oncologists haven’t had many tools other than traditional chemotherapy drugs to treat patients. That improvement would extend life by two to three months at best. Now for the first time we have a different pathway, the immunological pathway, and we have the tools to make it work.” Agenus actually had the first approved cancer vaccine, Oncophage, but that was in Russia to treat kidney cancer patients post surgically and will not be submitted for U.S. approval, he said.

Several other New England companies also are working on cancer drugs, including ONCoPEP Inc. of North Andover, which is developing a vaccine for smoldering myeloma (a precursor to multiple myeloma). Celldex Therapeutics Inc. (formerly Avant Immunotherapeutics Inc.), is making a brain tumor vaccine. InCytu’s vaccines are based on intellectual property from Harvard University.

In addition, RNAi-focused biotech firm Alnylam Pharmaceuticals Inc. of Cambridge has a license deal with Australia’s University of Queensland that gives the company access to RNAi intellectual property to develop cancer vaccines. And, RXi Pharmaceuticals Corp. of Worcester presented positive Phase 2 efficacy results at ASCO for its NeuVax peptide vaccine combined with Genentech/Roche’s Herceptin for breast cancer patients. The group of patients treated with the vaccine showed no cancer recurrence for three years, compared to 22 percent recurrence in patients receiving standard care.

BioVex Group Inc. of Woburn perhaps punctuated the value now being placed on cancer vaccine technology when it said in January that it would be acquired by biotech giant Amgen Inc. of Thousand Oaks, CA, for up to $1 billion. The transaction was completed March 4. The company is in a Phase 3 multinational study in metastatic melanoma and a Phase 3 study in squamous cell carcinoma of the head and neck with an oncolytic vaccine called OncoVEX(GM-CSF). The vaccine uses a virus to target and destroy certain cancer cells as well as create an immune response to tackle cancer cells throughout the body.

Melanoma was a hot topic at the ASCO meeting, because the cancer has been tough to arrest. Armen of Agenus said Yervoy in particular was a breakthrough, because it removes the braking system of the immune system and it also potentially could be used for other cancers including lung and prostate.

Bristol-Myers released results at ASCO of a second Phase 3 randomized trial of Yervoy showing it prolonged the lives of patients with metastatic melanoma. The FDA approved Yervoy for patents with unresectable or metastatic melanoma in March 2011.

The Agenus drug for GBM induces an immune response that lets patients fight their own disease, said University of California at San Francisco (UCSF) neurosurgeon Andrew Parsa, MD, who led the G-200 study. Without treatment, he said half of the patients in the trial would have died of the cancer within five to nine months based on historical data. After receiving the vaccine, the median survival for 30 patients who received at least four vaccinations was 11 months. Several have survived for more than a year.

A second objective of the trial was to see if the vaccine could produce an immune response in all patients, and the results showed that it did in every patient in the trial tested to date. This suggests that doctors may be able to extend survival even longer by combining the vaccine with other drugs that enhance this immune response, Parsa said. Some 17,000 Americans are diagnosed with glioblastoma every year, he said, and only 2 percent of them survive longer than five years, even with treatment. Based on the study results, Agenus has started working with UCSF and other experts to design a randomized trial.

“With the recent FDA approvals of Provenge and Yervoy, which both harness the power of the immune system to fight cancer and offer the potential for combination use with other immunological agents, I believe we are entering a new era in the treatment of cancer that could see substantially improved survival rates in patients fighting this disease,” Marcel Rozencweig, MD, acting chief medical officer of Agenus, said in a statement when the data was released.

The future may involve trials combining G-200 with other agents. “G-200 patients aren’t responding to anything. Everything else has failed,” Armen said. “People don’t live with this disease.” He said cancer vaccines have the potential to be the Holy Grail in the treatment of cancer, but for that promise to be fulfilled they must be able to direct the immune system to target a patient’s own cancer and their potency must be improved. Cancer vaccines combined with immune/pathogenesis modulators could enhance activity in late-stage cancers, and G-200 could be combined with Yervoy or other drugs.

Davis of Celldex takes it a step further. “The next generation of vaccines will have multiple targets in them in the same patient,” he said. Celldex’s lead candidate is a vaccine for EGFRvIII-expressing tumors. The company also uses monoclonal antibodies to target dendritic cells, which in turn spark an immune response.

Doris Peterkin, CEO of ONCoPEP (see sidebar), said patients are getting diagnosed earlier and living longer. “There’s a general acceptance in oncology of the value of immunotherapy, but the frustration is how to make it work. We need to understand why certain patients respond and others don’t,” she said. “One drug doesn’t work the same for every patient; it will be the same thing for immunotherapy.” She added that cancer vaccines have improved by leaps and bounds in the past two years, but it’s important to get to an earlier stage of the disease.

Added Davis, “In the first 30 to 40 years of the war on cancer, the most progress was made in understanding what is going on with cancer and the immune system. It will still be many decades before we say we can cure most cancers.” 

New Strategies used to Identify Changes in Head and Neck Cancers

Source: GenomeWeb Daily News
By Andrea Anderson

CHICAGO– Researchers are making progress using high-throughput strategies to find previously unappreciated genetic and epigenetic quirks in head and neck cancer — including changes that may prove useful for diagnosing and tracking disease.

Johns Hopkins University head and neck cancer research director David Sidransky described some of the work during an education session on molecular biology, targets, and pathways involved in head and neck cancer at the American Society of Clinical Oncology annual meeting here yesterday.

Speaking during the same session, JHU oncologist Christine Chung and the University of Chicago’s Ezra Cohen touched on strategies for targeting the types of mutations previously reported in head and neck cancer and the rationale behind targeted therapeutics already being tested or considered for the disease, respectively.

Past studies have uncovered muted DNA methylation across the genomes of several cancer types, Sidransky explained, though methylation is also bumped up at specific sites in certain tumor types.

Consequently, he said, researchers are using strategies such as real-time quantitative methylation-specific PCR (real-time QMSP) to look at methylation shifts in head and neck squamous cell carcinoma.

In particular, he described work comparing methylation patterns in saliva and serum samples from individuals with HNSCC to those in samples from more than 800 apparently healthy individuals who are considered ‘at-risk’ of the disease because of smoking status and other exposures.

At least two genes — KIF1A and EDNRB — seem to be more highly methylated in samples from those with HNSCC than at-risk control individuals, Sidransky said, suggesting methylation of these genes might prove useful for diagnosing and monitoring the disease.
Researchers are also finding mutations and rearrangements in several genes that appear to be promising biomarker candidates for distinguishing between HNSCC cases and controls using DNA from blood samples.

Moreover, Sidransky outlined some preliminary findings from exome sequencing studies of 32 head and neck cancer samples, followed by validation in another 88 samples.

Consistent with previous findings for HNSCC, he and his collaborators have found mutations in TP53 in nearly half of the tumors tested, Sidransky said.

But they are also seeing mutations in several other genes not previously linked to the disease, including NOTCH1 — a gene that’s known to be mutated in leukemia and other types of cancer. While NOTCH1 mutations are often activating in other cancers, he noted, results so far hint that the gene is frequently inactivated in HNSCC.

In addition, findings from the exome sequencing study point to genetic and epigenetic differences in HNSCC tumors that correspond to the human papilloma virus status. For example, Sidransky said, HPV positive tumors each tend to have fewer mutations overall compared to HPV negative tumors, which often come from smokers.

At the American Association for Cancer Research annual meeting in Orlando earlier this year, researchers from the Broad Institute and the University of Pittsburgh reported on findings from their own genome and exome sequencing studies of head and neck cancer, including whole exome data for 28 tumor-normal pairs and whole genome data for two tumor-normal pairs.

That team identified frequent mutations in TP53, HRAS, PIK3CA, and PTEN, along with somatic rearrangements and recurrent mutations in additional oncogenic pathways.

This news story was resourced by the Oral Cancer Foundation, and vetted for appropriateness and accuracy.

Soy increases radiation’s ability to kill lung cancer cells, study shows

Source: www.eurekalert.org/
Author: press release

Soy isoflavones block cancer cells’ DNA repair mechanisms while protecting normal tissue

A component in soybeans increases radiation’s ability to kill lung cancer cells, according to a study published in the April issue of the Journal of Thoracic Oncology, the official monthly journal of the International Association for the Study of Lung Cancer.

“To improve radiotherapy for lung cancer cells, we are studying the potential of natural non-toxic components of soybeans, called soy isoflavones, to augment the effect of radiation against the tumor cells and at the same time protect normal lung against radiation injury,” said Dr. Gilda Hillman, an associate professor in the Department of Radiation Oncology at Wayne State University’s School of Medicine and the Karmanos Cancer Institute in Detroit.

“These natural soy isoflavones can sensitize cancer cells to the effects of radiotherapy, by inhibiting survival mechanisms which cancer cells activate to protect themselves,” Hillman said. “At the same time, soy isoflavones can also act as antioxidants in normal tissues, which protect them against unintended damage from the radiotherapy. In a recent study, published in the Journal of Thoracic Oncology, we demonstrated that soy isoflavones increase killing of cancer cells by radiation via blocking DNA repair mechanisms, which are turned on by the cancer cells to survive the damage caused by radiation.”

Human A549 non-small cell lung cancer (NSCLC) cells that were treated with soy isoflavones before radiation showed more DNA damage and less repair activity than cells that received only radiation.

Researchers used a formulation consisting of the three main isoflavones found in soybeans, including genistein, daidzein and glycitein.

Previously, researchers had found that pure genistein demonstrated antitumor activity in human NSCLC cell lines and enhanced the effects of EGFR-tyrosine kinase inhibitors. This study showed that the soy mixture had an even greater antitumor effect than pure genistein. The soy mixture also is consistent with the soy isoflavone pills used in clinical studies, which have been proven to be safe, researchers said.

1. The study was supported by the American Institute for Cancer Research.
2. The Journal of Thoracic Oncology (JTO) is the official monthly journal of the International Association for the Study of Lung Cancer (IASLC). It is a prized resource for medical specialists and scientists who focus on the detection, prevention, diagnosis and treatment of lung cancer. It emphasizes a multidisciplinary approach, including original research (clinical trials and translational or basic research), reviews and opinion pieces.

April, 2011|Oral Cancer News|

Oropharyngeal cancer epidemic and human papillomavirus

Source: Foodconsumer
Author: Torbjörn Ramqvist and Tina Dalianis

A growing body of research shows that human papillomavirus (HPV) is a common and increasing cause of oropharyngeal squamous cell carcinoma (OSCC). Thus, the International Agency for Research against Cancer has acknowledged HPV as a risk factor for OSCC, in addition to smoking and alcohol consumption. Recently, in Finland, the United Kingdom, the Netherlands, the United States, and Sweden, incidence of OSCC has increased, and an increase in the proportion of HPV-positive tumors was noted. On the basis of these data and reports indicating that patients with HPV-positive cancer have their first sexual experience at a young age and have multiple partners, we postulate that increased incidence of OSCC in the United States and some countries in northern Europe is because of a new, primarily sexually transmitted HPV epidemic. We also suggest that individualized treatment modalities and preventive vaccination should be further explored.

In many countries, vaccines against some human papillomavirus (HPV) types are now administered to girls and young women with the goal of protecting them against HPV-induced cervical cancer (1,2). The introduction of HPV vaccines has also drawn more attention to the fact that HPV is associated not only with cervical cancer and genital warts but also with other tumors, such as head neck and anogenital cancers (3). We focus on the role of HPV in the increased incidence of oropharyngeal squamous cell carcinoma (OSCC), the head and neck cancer in which HPV is most commonly found (4).

Head and neck cancer most commonly is of the squamous cell carcinoma type (HNSCC) and includes cancers of the oral cavity, oropharynx, hypopharynx, larynx, sinonasal tract, and nasopharynx. HNSCC is the sixth most common type of cancer in the world; almost 600,000 cases are reported annually, and of these, ≈10% (or more for some geographic locations) are OSCC (5). Globally, the incidence and localization of HNSCC varies widely. It is the most common form of cancer in India, and incidence is higher in countries in Latin America than in the United States and northern Europe. In addition, men are generally more often affected than women. Smoking, alcohol consumption, and betel chewing are traditional risk factors for HNSCC and OSCC (6). However, during the past decade several reports have documented HPV in OSCC (7–9). HPV infection, with dominance of HPV16 infection, has therefore been acknowledged by the International Agency for Research against Cancer as a risk factor for OSCC (10). Moreover, there are accumulating reports from many countries that the incidence of OSCC is increasing. We suggest that this increase is caused by a slow epidemic of HPV infection–induced OSCC.


Tonsillar cancer is the most common OSCC, followed by base of tongue cancer. Together, these 2 cancers account for 90% of all OSCCs (6,9). Patients usually do not seek counseling until the tumors are large because small tumors cause little distress and may not be noticed by the patient. Curative treatment implies surgery, radiotherapy, and chemotherapy; the goal is to cause as little functional and cosmetic damage as possible (6,9). If a cure cannot be obtained, palliative therapy is given to treat pain and discomfort. Similar to HNSCC, in general, survival rates for patients with OSCC are poor. Patients with OSCC have an overall 5-year survival rate of ≈25% (6,9). Furthermore, even when standardized treatment is used and tumors are at the same stage and have similar histologic features, it is difficult to predict the outcome. Several reports now describe the incidence of OSCC as increasing and indicate that HPV-positive OSCC has a better clinical outcome than HPV-negative OSCC (7–9,11–19). Thus, predictive and prognostic markers would be of clinical value for prevention and treatment of OSCC.


There are >100 HPV types, some found in skin warts and others in mucous tissues, and the association of different HPV types with cervical, some anogenital, and head and neck cancers is well established (3). The 8-kb, double-stranded, circular DNA HPV genome, enclosed in a 52–55 nm viral capsid, codes for the L1 and L2 viral capsid proteins and for the E1–E2 and E4–E7 proteins, which play major roles in gene regulation, replication, pathogenesis, and transformation (3). In high-risk HPVs (i.e., those that are more likely to cause lesions that may develop into cancer [www.cancer.gov/cancertopics/factsheet/Risk/HPV]), E6 and E7 deregulate cell cycle control by E6 binding and degradation of p53, and E7 binds and inhibits the function of the retinoblastoma protein (Rb) (3). The L1 protein can self-assemble into virus-like particles, which form the basis of both currently approved vaccines against HPV infection (1–3).

HPV and Methods for Detection in OSCC

During the past few decades, HPV DNA has been detected in ≈25% of HNSCCs overall, but especially in OSCC, for which 45%–100% cases were reported to be HPV positive (7–9,11–19). The latter variation may depend on OSCC location, the type of specimens available, the techniques used for testing, and the time period and country from which the sample material was obtained (7–9,11–19).

Analysis of HPV DNA was (and still is) performed primarily by using formalin-fixed, paraffin-embedded tissue, in which the DNA can be partially degraded. It is now widely accepted that it is easier to detect longer HPV DNA fragments in fresh or fresh-frozen material, although newer techniques are more sensitive. Many early studies during the 1980s were based on Southern blot techniques or in situ hybridization for detection of HPV.

Since the 1990s, virology laboratories used PCR for detection of HPV DNA (20–23). Screening for HPV was initially performed by using general PCR primers for HPV, which enabled detection of several HPV types (21–23). PCR of a control cellular gene was used to assess the DNA quality of samples. These techniques are robust and are still used but need additional methods for HPV typing. There are now many other methods that directly determine the presence of several different HPV types. The Food and Drug Administration–approved Hybrid Capture II (Digene Corporation, Gaithersburg, MD, USA) detects 5 low-risk and 13 high-risk HPV types and uses the fact that HPV DNA hybridizes with synthetic RNA probes complementary to DNA sequences from specific HPV types (20). An assay used in several studies, the Roche (Basel, Switzerland) linear array HPV Genotyping Test, detects 37 HPV types and is based on a method developed by Gravitt et al. (24). In this method, HPV PCR products are hybridized to a linear array of type specific probes. Recently, Schmitt et al developed a sensitive bead-based multiplex method, originally set up for 22 different HPV types but later expanded, in which HPV PCR products are coupled to type-specific probes on beads and analyzed by using Luminex (25).

To assay for biologic activity of HPV in tumors, analysis of E6 and E7 expression by detecting E6 and E7 mRNA by reverse transcription followed by real-time PCR is also often performed (13). In pathology departments, HPV screening is often conducted by in situ hybridization, and in some instances p16 immunohistochemical analysis is used as a substitute to assay for biologically active HPV because there is a correlation between the presence of HPV and overexpression of p16 (26,27).


When HPV in OSCC became more obvious, several studies concentrated on characterizing HPV-positive OSCC (4,7–9,11–19). HPV type 16 was highly prevalent (≈90%) in OSCC in all studies; other HPV types (e.g., HPV-31, -33, -58, -59, -62, and -72) were less common, and HPV was demonstrated to be episomal or integrated into the cellular genome (14,28).

In several studies, E6 and E7 expression in OSCC were shown, suggesting that HPV was actively involved in the etiology of the tumors (13). In addition, the association of p16 overexpression with HPV was a further indicator of active E7 because of E7-induced cell cycle activation and upregulation of p16 by inactivating the Rb pathway (3,26,27). HPV-positive tumors were also less likely to have mutated p53 (7) and were more frequently aneuploid and less differentiated than HPV-negative tumors (29). Furthermore, comparative genomic hybridization indicated that HPV-positive tonsillar cancer, in contrast to HPV-negative cancer, often showed chromosome 3q amplification similar to that in HPV-positive cervical and vulvar cancer, which further supports the oncogenic role of HPV in OSCC (30).

It was also observed that patients with HPV-positive OSCC were younger and lacked the traditional risk factors of smoking and alcohol consumption (7–9,13,17). Moreover, a major feature, noted in several studies, was that HPV was a favorable prognostic factor for clinical outcome of OSCC, as demonstrated in Figure 1 (7–9,11–19). This finding was independent of tumor stage, age, gender, grade of differentiation, p53 immunohistochemical results, or DNA ploidy (7–9). However, Lindquist et al. (13) observed that patients with HPV-positive tonsillar cancer who had never smoked had a better prognosis than those who were smokers, and this observation was recently confirmed by Ang et al. (16). The reasons for this finding are most likely complex and should be investigated further. One could speculate, for example, that HPV induces an immune response and that smoking abrogates this response. A different option is that smoking and HPV in combination induce a different category of tumors and that smoking induces additional genetic alterations in these tumors, as was also suggested by Ang et al. (16).

In general, the prognostic value of HPV status is for OSCC and not for HNSCC. In some studies with conflicting findings, the prognostic value of HPV was determined for all HNSCC anatomic sites (9). However, because there are differences in survival rates and presence of HPV at different locations, even for OSCC, studies should be performed per location.

In summary, the accumulated data suggest different entities of OSCC, where some primarily depend on smoking and alcohol and others on HPV infection. It is also likely that there are combined etiologies. Nevertheless, patients with HPV-positive OSCC consistently have a better prognosis (7–9,11–19).

An HPV-induced Epidemic of OSCC

We suggest the increased incidence of OSCC depends on HPV infection and results in an increased proportion of HPV-positive OSCCs. During the past decades, studies from the United States, Finland, Sweden, the Netherlands, the United Kingdom, and Scotland showed an increase in the incidence of OSCC, tonsillar cancer, and base of tongue cancer (31–36). In addition, during the past 10 years, an increase in the proportion of HPV-positive OSCC has been reported, and we speculate that this is not caused simply by use of more sensitive diagnostic techniques. In many of these studies the same assay was used when studying OSCC over time (12,18,19). Furthermore, the general PCR amplifiability of the DNA from the older tested samples was also validated.

Using the Swedish Cancer Registry, which covers basically all cancer cases in Sweden for 1970–2002, we disclosed a 2.8-fold increase (2.6-fold for men and 3.5-fold for women) in the incidence of tonsillar cancer in the Stockholm area (12), where 25%–30% of all patients in Sweden with tonsillar cancer are treated. In parallel, we examined all 237 available samples from the 515 patients with tonsillar cancer in Stockholm during the same period and found a 2.9-fold increase in the proportion of HPV-positive tonsillar cancer from 23% to 68% (12). We thus suggested HPV infection played a role in the increase of this disease (12).

In continuation of the above study, we followed the incidence of tonsillar and base of tongue cancer in Stockholm in the Swedish Cancer Registry and demonstrated a substantial increase for both tumor types during 1970–2006, as shown in Figure 2 (18,19,34). We then performed a follow-up study in the Stockholm area of the prevalence of HPV in tonsillar cancer during 2003–2007; using the Swedish Cancer Registry, we identified 120 patients (18). Using the same methods as in the first study, we found that the proportion of HPV-positive cancers in the 98 available pretreatment biopsy specimens had significantly increased both from 1970 through 2007 (p<0.0001) and from 2000 through 2007 (p<0.01). During the last 2 years of the study (2006–2007), 93% of all tonsillar cancer was HPV positive. Moreover, the incidence of HPV-positive tumors almost doubled each decade during 1970–2007, indicating a 7-fold increase over the whole period; in parallel, a decline of HPV-negative tumors was observed (Figure 3).

Shortly afterwards, we demonstrated that the prevalence of HPV-positive base of tongue cancer also had increased in the Stockholm area during 1998–2007 (19). When we analyzed 95 pretreatment biopsy specimens from base of tongue SCC from the 109 patients reported during 1998–2007 in the Swedish Cancer Registry in a similar way as above, we found an increase in the proportion of HPV-positive tumors from 54% in 1998–1999 to 84% in 2006–2007.

The strength of the above studies is that the Swedish Cancer Registry covers all cancer cases in Sweden and that we have analyzed all available pretreatment diagnostic biopsy specimens for HPV. The limitation of our study is that Sweden is a small country with only 9 million inhabitants.

Covering almost the same time period as above, Chaturvedi et al. reported an increase in the proportion of potentially HPV-related OSCC in the United States (35). However, the previous year, Sturgis and Cinciripini had already proposed a possible emerging epidemic of HPV-associated cancers (11). More recently, Marur et al. reviewed many studies, further supporting an increase in OSCC (17). Notably, it was also suggested that the increase in OSCC occurs mainly in men (17). However, using the Swedish Cancer Registry, in the Stockholm area, Hammarstedt et al. observed also an increase of OSCC in women (12). It is necessary to acknowledge that the numbers of women with OSCC are more limited and that it could be more difficult to identify major changes in this group.

The possible causes for this increase have been discussed extensively and have focused on changes in sexual patterns, such as increased oral sex or increasing numbers of sex partners. A significant association has been shown between HPV-positive tonsillar cancer and early initial sex or number of oral or vaginal sex partners (37).

Furthermore, in a recent study by D’Souza et al., it was shown that the risk of developing oral HPV infection increased with increases in lifetime oral or vaginal sex partners (38). It has also been reported that not only oral sex, but also open-mouthed kissing, was associated to the development of oral HPV infection (38). In this study, 2 study populations were included, one (332 patients) consisted of a control patient group >18 years of age from the Johns Hopkins outpatient otolaryngology clinic (2000–2006) enrolled in 2 case–control studies within a prospective cohort with HNSCC. The other (210 students) consisted of students >17 years of age recruited from the campuses of Towson University and the University of Maryland in 2007. The latter study may have had some limitations because it was not population based, and data for open-mouthed kissing for control patients and smoking for college students were absent. Nevertheless, this study suggests that oral-to-oral contact may play a role for oral HPV transmission and could play a major role in timing of prophylactic vaccination of children.

Several reports show an increase in OSCC and the proportion of HPV-positive OSCC and an association of the latter to early sex debut and many partners. Thus, we suggest that we are encountering a slow epidemic of mainly sexually transmitted HPV-induced OSCC.

HPV in OSCC and Consequences for Treatment and Prevention

The possibility that we are dealing with an HPV-induced epidemic of OSCC warrants special attention. For example, in Stockholm, the incidence of HPV-positive tonsillar cancer has increased 7-fold over 30 years (18). OSCC now accounts for approximately one third of all HNSCC cases annually in Sweden. The fact that HNSCC in general is decreasing, and OSCC is increasing, may in 10 years result in OSCC accounting for half of all HNSCCs in Sweden, and similar trends are likely elsewhere, e.g., the United States, United Kingdom, the Netherlands, and Finland. It is also known that patients with HPV-positive OSCC are younger and have a better prognosis than HNSCC patients and patients with HPV-negative OSCC (7,8). In contrast, because of the poor prognosis for HNSCC and, in the past for OSCC, therapeutic measures have recently been intensified with induction chemotherapy, hyperfractionated radiotherapy, surgery, and occasional use of epidermal growth factor receptor inhibitors. This intensified therapy results in more severe acute and chronic side effects, such as difficulties in swallowing or talking, dry mouth, and necrosis of the jawbone, and is also more expensive for society. Accordingly, it is possible that increasing numbers of OSCC patients with a better prognosis are being treated with intensified therapy. As a result, many patients have substantial chronic unnecessary side effects. It is therefore necessary to identify which patients need and which do not need intensified treatment, both to increase patient survival times and quality of life and for the socioeconomic benefit of society.

Several reports have been published and other studies are ongoing to assess which molecular factors, such as p16, p53, and others, besides the presence of HPV in OSCC, can best predict clinical outcome and which treatments are optimal according to the same predictive markers (7–9,17,26). Some retrospective reports have suggested that persons with HPV-positive OSCC have higher response rates to chemotherapy and radiation; however, in other reports this has not been confirmed (9,17). A recent study also observed that tumor HPV status is a strong independent prognostic factor for survival among patients with HPV-positive OSCC irrespective of treatment (16). However, in the same study, among patients with HPV-positive tumors, the risk for death significantly increased with each additional pack-year of smoking, independent of treatment modality (16), a result similar to that found by Lindquist et al. (13).

It has been shown in an experimental setting that HPV-positive tumors were not more curable on the basis of increased epithelial sensitivity to cisplatin or radiation therapy (39). Instead, Spanos et al. demonstrated that radiation and cisplatin induced an immune response to this antigenic type of cancer. This finding could suggest that the presence of HPV in a tumor induced by smoking could be of benefit, but it is possible that smoking also may abrogate the immune response. As mentioned, the relationship between smoking and HPV and their roles in OSCC is most likely complex. In future studies, it would therefore be valuable to obtain more molecular and immunologic information and to determine if it is a survival benefit to stop smoking during and after therapy.

Summarizing treatment of OSCC patients, it is obvious that additional information will be required before it will be possible to guide treatment decisions for the individual patient on the basis of HPV status. Nevertheless, there is accumulating evidence that HPV status and overexpression of p16, and having never smoked, is of benefit. Future prospective clinical studies, including diagnostics of HPV, molecular and immunologic profiles, history of smoking, cessation of smoking during therapy, and effects of different treatment modalities and their side effects on quality of life, will be of benefit for personalized treatment.

Finally, it is also essential to keep in mind that we now have vaccines directed against HPV16, which accounts for ≈80%–90% of all HPV-positive OSCC, at least in Europe and the United States (7–9,11–9). Although it will likely take several decades before the effects of HPV vaccination on cancer incidence will be detected, it is crucial to monitor the effects of the present HPV vaccination, not only on the incidence of cervical cancer but also on the incidence of OSCC.

Few if any of other studies have focused on performing isolated health economic analysis of the effect of HPV on OSCC. However, a recent study pointed out that there is an improvement of the present cost-effectiveness of HPV vaccines when the effects on other HPV-associated tumors cancers are included (40). Furthermore, in countries with effective cervical cancer screening programs, other HPV-associated noncervical cancers represent a relatively high proportion of HPV-positive cancers (15). Considering that OSCC is the second most common HPV-associated cancer and its incidence is increasing, the effect of the HPV vaccine on this tumor deserves attention, and we need to know if future vaccination against HPV infection should include both women and men.

This work was supported in part by the Swedish Research Council, the Swedish Cancer Foundation, the Stockholm Cancer Society, the Stockholm City Council, and the Karolinska Institutet, Sweden.

Dr Ramqvist is a senior researcher and associate professor at the Karolinska Institutet. His research interests are murine and human polyomaviruses and human papillomaviruses.

Dr Dalianis is a specialist in clinical virology and immunology and a professor in tumor virology at the Karolinska Institutet. Her research interests are human papillomaviruses and murine and human polyomaviruses.


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November, 2010|Oral Cancer News|

Cancer answer? Researchers are working on a more individual approach to each tumour

Source: macleans.ca
By: Kate Lanau

This summer, Vancouver cancer researchers announced a medical first. Presented with an extremely rare case of tongue cancer—it was so unusual there were no standard treatments to use—they sequenced the DNA of the patient’s tumour, and discovered similarities with another cancer (renal cell carcinoma, a type of kidney cancer) for which there’s a known therapy. The patient received drugs tailored to these results, and the cancer stopped growing for several months. Steven Jones, a molecular biologist with the B.C. Cancer Agency Genome Sciences Centre and one of two lead researchers on the study, calls it a breakthrough. It isn’t standard in hospitals to genetically sequence a patient’s tumour, but “the goal would be, maybe in 10 years, this would be routine,” he says.

Dr. Leif Ellisen, an associate professor of medicine at Harvard Medical School, is working to bring tumour genotyping from the lab into the clinic. He and a team have designed a system that can screen relatively large numbers of patients for a variety of mutations across different cancer genes. These genetic mutations are a tumour’s “Achilles’ heel,” noted a recent editorial in the journal EMBO Molecular Medicine. “Every tumour has a flaw,” says Ellisen, who’ll be discussing his work as part of the Scienta Health Series in Toronto on Oct. 7, and his goal is to find it.

It’s the mantra of a growing number of researchers, who tout personalized medicine—treatments tailored to each individual—as the future of cancer care. Traditionally, cancer treatment “has been one-size-fits all,” Ellisen says. “If it’s breast cancer, you treat it one way; if it’s lung cancer, you treat it another.” The downside is that costly drugs are administered to patients, sometimes with harmful side effects and no real promise they’ll work. “The treatment needs to be tailored to the individual characteristics of the patient and, we’re learning now, the characteristics of the tumour,” he says. Cancers are typically classified by the organs where they arise, but it’s possible that a breast cancer and a lung cancer, for example, might share a genetic abnormality. As a result, they might even respond to the same treatment.

That’s the concept behind smart drugs, which are being developed to target specific molecular pathways activated by cancer gene mutations. “These drugs work in a very specific way, as opposed to chemotherapy, which works in a general way,” Ellisen says. One example is Herceptin, a drug that treats certain types of breast cancer. Herceptin inhibits a gene amplified in some women, but not others, that acts like a growth factor for cancer cells. Now, when breast cancer patients are diagnosed, “one of the first tests done on the tumour is to look for this gene,” called HER2, Ellisen says. “If it’s activated, the patient will get Herceptin. If not, they won’t, because it wouldn’t benefit them.”

A broader test might pick up on mutations we wouldn’t expect to see in a given tumour, but which might have a known treatment available. “We needed to develop a technology to look across the spectrum of cancer genes in the tumours,” Ellisen says. “That’s the only way we could personalize the therapy.” The genotyping system they developed can test for over 150 mutations in 18 cancer genes. In a journal, Ellison and his fellow researchers said they opted to scan for “cancer mutations most likely to have immediate clinical impact,” either because they’re already targeted by FDA-approved drugs, or new drugs in development.

Genotyping tumours in the lab is one thing, but in a hospital setting with actual patients, it’s quite different.

“One of the biggest challenges was purifying and analyzing genetic material” in a quick and reproducible fashion, Ellisen says. Tumour samples are generally “set in fixatives, embedded in wax blocks, and stored at room temperature,” he says, and the specimens themselves can have impurities, with tumour cells mixed in with normal ones. All this can, in some cases, make it much harder to detect mutations. Thanks to a series of robots the team designed, the lab can handle complete snapshot genotyping of up to 50 samples a week, “a substantial fraction of all the cancers diagnosed at Mass Gen,” says Ellisen, co-executive director of the hospital’s Cancer Center’s Translational Research Laboratory, which is focused on personalized cancer care.

Ellisen acknowledges we’ve still got a lot to learn. “We don’t know all the cancer genes that exist,” he says, and we need more and better drugs to treat them. (The sequence of the first cancer genome was published just two years ago.) But the field is advancing in leaps and bounds. In April, the International Cancer Genome Consortium, made up of several countries including Canada and the U.S., announced a plan to map the genomes of 25,000 cancer samples. (Canadian researchers are focused on prostate and pancreatic tumours, while the U.S. is working on brain, lung and others.) “There’s been a million-fold improvement in sequencing technology since 2001,” says Dr. Tom Hudson, president and scientific director of the Ontario Institute for Cancer Research. Even so, he expects it will take up to 10 years to complete it.

As we learn more about cancer genes, Ellisen’s system can be updated. “This platform is very scalable, so we can add mutations as new discoveries are made,” he says. Because it’s relatively simple, it could be adopted in clinics elsewhere, he believes. The cost of a test is similar to an MRI scan. Meanwhile, other cancer centres are trying out different genotyping systems, and sharing information about what works best.

The day when cancer patients can be routinely treated based on the genetic makeup of their tumours is still a ways off.

As Ellisen notes, there’s “only a relatively small number of effective targeted therapies available for routine clinical use,” although that number is growing. As we continue to unravel the genetic mutations that lead to cancer, and develop smart drugs to target them, treatment will change drastically, he and others predict.

“There’s no question that this is going to be the way cancer is approached in the future,” Ellisen says.

October, 2010|Oral Cancer News|

High-risk human papillomavirus in esophageal squamous cell carcinoma

Source: cebp.aacrjournals.org
Authors: Annika Antonsson et al

Although most cases of esophageal squamous cell carcinoma (ESCC) in western populations have been attributed to high levels of exposure to tobacco and alcohol, infectious agents have been postulated as possible causes, particularly human papillomavirus (HPV).

To explore this issue, we analyzed HPV DNA prevalence and HPV types together with lifestyle factors, in relation to tumor stage and survival in a low-incidence population. Archived tumor samples from a nationwide cohort of 222 ESCC patients were tested for the presence of HPV DNA by PCR; positive samples were sequenced to determine HPV type, and p16INK4a status was assessed by immunohistochemistry.

Of 222 ESCC patients, 8 tested HPV positive (prevalence, 3.6%; 95% confidence interval, 1.1-6.1%), of which 6 were HPV-16 positive and 2 were HPV-35 positive. Four of the eight HPV-positive tumors overexpressed p16INK4a. None of 55 normal esophageal tissue samples from healthy participants had any detectable HPV. Although the numbers were low, it seemed that patients with HPV-positive ESCC tumors were younger than those with HPV-negative tumors (mean age, 60.8 versus 65.3 years, P = 0.18) and had higher body mass index (BMI) throughout life (mean current BMI of 25.1 for HPV positive, 22.2 for HPV negative, P = 0.08; mean BMI at 20 years of 25.8 for HPV positive, 22.1 for HPV negative, P = 0.003). We found no difference between patients with HPV-positive and HPV-negative tumors with respect to other lifestyle factors.

Conclusions: These findings suggest a very low prevalence of HPV DNA in human ESCC.

HPV is very unlikely to be a common cause of ESCC in Australia. Cancer Epidemiol Biomarkers Prev; 19(8); 20.

1. The Australian Cancer Study: Esophageal Cancer: Investigators: David C. Whiteman, Penelope M. Webb, Adele C. Green, Nicholas K. Hayward, Peter G. Parsons, David M. Purdie. Clinical collaborators: B. Mark Smithers, David Gotley, Andrew Clouston, Ian Brown. Project Manager: Suzanne Moore. Database: Karen Harrap, Troy Sadkowski. Research Nurses: Suzanne O’Brien, Ellen Minehan, Deborah Roffe, Sue O’Keefe, Suzanne Lipshut, Gabby Connor, Hayley Berry, Frances Walker, Teresa Barnes, Janine Thomas, Linda Terry, Michael Connard, Leanne Bowes, MaryRose Malt, Jo White.

2. Study of Digestive Health: Investigators: Queensland Institute of Medical Research, Brisbane Australia: David C. Whiteman, Adele C. Green, Nicholas K. Hayward, Peter G. Parsons, Sandra J. Pavey, David M. Purdie, Penelope M. Webb; University of Queensland, Brisbane, Australia: David Gotley, B. Mark Smithers; The University of Adelaide, Adelaide, Australia: Glyn G. Jamieson; Flinders University, Adelaide, Australia: Paul Drew, David I. Watson; Envoi Pathology, Brisbane, Australia: Andrew Clouston.

3. Research Staff: Project Manager: Suzanne O’Brien; Research Scientist: Derek Nancarrow; Research nurses: Andrea McMurtrie, Linda Terry, Michael Connard, Deborah Roffe, Lorelle Smith, Marian Martin, Jeanette Mayhew, Susan Perry, Marcia Davis.

Annika Antonsson1, Derek J. Nancarrow2, Ian S. Brown3, Adele C. Green1, Paul A. Drew4, David I. Watson5, Nicholas K. Hayward2 and David C. Whiteman1 for the Australian Cancer Study1

Authors’ affiliations:
1Genetics and Population Health Division and 2Oncogenomics, Queensland Institute of Medical Research, and 3Sullivan Nicolaides Pathology and Royal Brisbane and Women’s Hospital Brisbane, Brisbane, Queensland, Australia; 4School of Nursing and Midwifery, and 5Department of Surgery, Flinders University, Bedford Park, South Australia, Australia

August, 2010|Oral Cancer News|