“When I arrived at Yale in 1983, people didn't think these viruses were important to cancer. At conferences the human papillomavirus was always the last talk of the meeting. Now it’s taken center stage.”
—Daniel DiMaio.

“Cervical cancer is the ideal cancer in which to demonstrate the principle of anticancer vaccines, because we know what the tumor antigens are.”
—Janet Brandsma

“It used to be a job to convince people that viruses were an important part of the cancer story. There had been a lot of research, but people just didn’t believe it. … Now people pretty much accept the idea.”
—George Miller

The virus behind the cancer

One in 10 human cancers starts with a viral infection, often the ubiquitous human papillomavirus. Yale scientists want to know why—and are hot on the trail of new vaccines and therapies to treat the virus behind the cancer.

By Jennifer Kaylin
Illustrations by Yasuo Tanaka
Photographs by Terry Dagradi

More than 50 years ago, a young woman named Henrietta Lacks was diagnosed with cervical cancer. Despite surgery and aggressive radiation therapy, the cancer soon spread throughout her body, and on October 4, 1951, she died.

It was a cruel death for the 31-year-old mother of five, but Lacks’ story didn’t end there. George O. Gey, M.D., head of tissue culture at Johns Hopkins University, where Lacks was treated, had been searching, for research purposes, for a line of human cells that could live indefinitely outside the body. He got his wish when cells from Lacks’ cancerous tumor were cultured. Just as they had done in her body, the cells multiplied ferociously in the lab, crawling up the sides of test tubes and consuming the medium around them. An entire generation of the cells reproduced every 24 hours.

Referring to Lacks’ cells, Gey declared at the time, “It is possible that, from a fundamental study such as this, we will be able to learn a way by which cancer can be completely wiped out.” To this day, Lacks’ cells, known as the HeLa cell line, are some of the most robust and rapidly growing cells known to science. They are still used by thousands of researchers around the world to decipher the complexities of cell biology, particularly as they apply to cancer.

At Yale, scientists are using the HeLa cell line to study, among other things, the human papillomavirus (HPV) that causes the cervical cancer that killed Lacks. “Her legacy,” says Daniel C. DiMaio, M.D., Ph.D., the Waldemar Von Zedtwitz Professor of Genetics and professor of therapeutic radiology, “is that her cells are helping us unravel the pathogenesis of cervical cancer, so that some day we might be able to prevent and treat it. It’s rather remarkable.”

The field of human tumor virology is still a relatively new area of scientific inquiry. Although it has been known for nearly a century that viruses can cause tumors in animals, only in recent decades have human tumor viruses been identified. Researchers at Yale, among them I. George Miller, M.D., have contributed to our understanding of the mechanisms of viral tumorigenesis.

Miller, the John F. Enders Professor of Pediatrics and professor of epidemiology and of molecular biophysics and biochemistry, was the first to show that a human virus can cause tumors in primates. Experiments he conducted at Yale in the 1960s showed that the Epstein-Barr virus (EBV) could cause lymphoma in cotton-top marmosets. He also showed that the virus was very effective at changing normal human lymphocytes into cells with properties of cancer cells in culture.

More recently, DiMaio’s lab demonstrated that cervical cancer cells need the viral proteins to grow, thus raising the possibility that the cancers can be treated with antiviral drugs. DiMaio, Janet L. Brandsma, Ph.D. ’81, and others are currently working on a vaccine to treat patients with cervical cancer.

Besides these advances, Yale researchers who specialize in tumor virology believe their work could have wider applications, potentially expanding knowledge of a range of cancers and other illnesses and biological processes, such as cellular aging. “It will help us understand all cancers,” says Brandsma, an associate professor of comparative medicine and pathology. “Most small mutations in cellular genes are very subtle, but with viral cancers, the viral genome in the cancer cell is foreign and easier to recognize. It’s an excellent model.”

Chickens, rabbits, warts and mice
More than 10 percent of all cancers in humans are strongly associated with infection by tumor viruses, and roughly 15 percent of all cancer deaths worldwide are caused by viruses. “It’s a very important problem,” DiMaio says. But he also sees tumor virology as a tremendous opportunity. “Once you know that a cancer is caused by a virus, you are far ahead of where you’d be for any other cancer, because you’ve identified the target, you’ve identified the cause and you have well-established ways to prevent or treat the disease that just don’t exist for spontaneously arising tumors.”

To say that certain viruses cause certain cancers can be misleading. You can’t catch cancer from another person, and most people who are infected with HPV, for example, won’t get cervical cancer. However, everyone who gets cervical cancer has the HPV infection. “Other things have to go wrong in order for the cancer to develop,” DiMaio explains, “but the virus contributes in an essential way. If you prevent virus infection by vaccination, you don’t get the cancer, and if you turn off the virus, the cancer can’t grow.”

HPV is the best-understood example of how a virus leads to cancer. Two things have to happen: First, viral gene products cause the cells to become genetically unstable and accumulate mutations that render cells unresponsive to aspects of growth control and the immune response. Second, the viral oncogenes provide a sustained stimulus to cell growth.

The first clue that there was a viral link to certain cancers came in 1911. Using a virus found in chickens, F. Peyton Rous, M.D., a scientist at the Rockefeller Institute for Medical Research, showed that the chicken sarcoma could be induced in other chickens. “There was a lot of doubt about what applicability it had, if any, to human disease,” says Miller. But in 1966 Rous shared the Nobel Prize in physiology or medicine for his research on the link between viruses and cancer, and the chicken virus became known as the Rous sarcoma virus.

Another important development, Miller says, came in the 1930s, when Richard Shope, M.D., one of Rous’ collaborators and the father of the late Yale epidemiologist Robert E. Shope, M.D., HS ’58, was out hunting with a friend. The friend mentioned that he’d seen rabbits with horns—actually giant warts. Shope asked his friend to send him some of the horns, which he then ground up, so he could isolate the virus causing the warts. When he injected the virus into other rabbits, they also grew horns. Interestingly, when New Zealand white rabbits were inoculated with the virus, they grew horns, but Shope couldn’t recover the virus; in cottontail rabbits, the virus was retrievable. This discovery raised the question of viral latency, which scientists now know is intrinsic to the behavior and biology of tumor viruses. (Miller is currently researching latency as it relates to the Kaposi sarcoma virus. He’s trying to determine what the suppressor mechanism is and why latent-state viral genomes are suppressed in the tumor cells and then periodically reactivated.)

In the early 1950s Ludwik Gross, M.D., head of cancer research at the Bronx (N.Y.) VA Hospital, opened the field of tumor virology in mammals with his discovery of what became known as the Gross mouse leukemia virus. Gross showed that a virus led to mouse leukemia and could be passed from one generation to the next.

Although these and other studies unequivocally showed that viruses can lead to tumors in animals, making the leap to human tumor viruses wasn’t easy. Researchers encountered several obstacles. For starters, only a small percentage of people who are infected actually develop cancer; it takes more than a virus infection for a tumor to form; and other factors, such as immunosuppression or exposure to another carcinogen, must be present. Finally, it can take decades for symptoms to appear.

Despite these challenges, in 1965 the first bona fide example of a human tumor virus—EBV—was discovered in cells from Burkitt lymphoma. Since then scientists have identified six viruses that have been shown to play a role in human cancers.

HPVs are a family of small DNA viruses that typically cause benign warts. However, certain high-risk HPV types have been linked to a variety of carcinomas, the most prevalent being cervical cancer. HPV is also thought to play a role in other anogenital cancers, skin cancers and some head and neck tumors.

Hepatitis B virus and hepatitis C virus are genetically unrelated, but both can cause acute and chronic liver disease, which, under certain conditions, can progress to primary hepatocellular carcinoma. EBV is a herpes virus that can cause mononucleosis. However, EBV has also been linked to Burkitt lymphoma and nasopharyngeal carcinoma, and it has been implicated in some forms of Hodgkin disease and gastric carcinoma. Human herpes virus 8 (HHV-8), also known as Kaposi sarcoma herpes virus, is related to EBV. It was first identified in the tumor DNA of a patient with Kaposi sarcoma, a rare tumor until the aids epidemic, when it became one of the most common causes of cancer deaths among aids patients. HHV-8 is also believed to play a role in Castleman disease and body cavity lymphoma. Finally, human T lymphotropic virus type 1 leads to a rare tumor, adult T-cell leukemia/lymphoma, in the Far East and the Caribbean basin, as well as to some nonneoplastic diseases.

“It used to be a job to convince people that viruses were an important part of the cancer story. There had been a lot of research, but people just didn’t believe it. They wondered, for example, why so many people who are infected don’t get cancer,” says Miller. “We had to fill in the details. Now people pretty much accept the idea.”

“When I arrived at Yale in 1983, people didn’t think these viruses were important to cancer,” DiMaio says. “At conferences the human papillomavirus was always the last talk of the meeting. Now it’s taken center stage.” That’s partly because, of all the viruses found to play an etiologic role in human cancers, the HPV types (16 and 18) linked to cervical cancer are probably the best-understood and the ones that hold the greatest promise for vaccines to be used for prevention and treatment.

Tight corsets and HPV
Early thinking on cervical cancer and what causes it would hardly suggest such a rosy scenario. In 1842 an Italian physician in Florence observed that married women in the city were getting cervical cancer, but nuns in nearby convents weren’t. Although this observation would seem to point to a link between sexual activity and cervical cancer, the physician did not make this connection. He also observed that nuns had higher rates of breast cancer, and suggested that the nuns’ corsets were too tight. “Clearly they had no clue,” DiMaio says, “but the observation was significant.”

Beginning in 1975, the virologist Harald zur Hausen, M.D., D.Sc., figured out what had eluded the Florentine physician. Zur Hausen, who for 20 years headed the German Cancer Research Center in Heidelberg, showed that HPV, a common infection spread through skin-to-skin contact and sex, could lead to cervical cancer. He and his research team successfully isolated several genotypes of the virus, some of which they linked to genital warts and others to cervical cancer.

Today, cervical cancer is responsible for 250,000 deaths each year worldwide, according to Charles J. Lockwood, M.D., the Anita O’Keefe Young Professor of Women’s Health and chair of the Department of Obstetrics, Gynecology and Reproductive Sciences. In the United States, where early screening has greatly reduced the mortality rate due to cervical cancer, about 5,000 women a year still die of the disease.

“From a mortality standpoint, the problem in this country is largely contained, but worldwide it’s a huge problem,” says Lockwood. “From a financial standpoint it remains a major problem in this country. The cost of surveillance and preventive treatments is astronomical ($200 million a year just for screening), and a woman who has multiple surgical treatments for precancerous conditions of the cervix, such as cone biopsies or loop electrocautery excision procedures, is at a higher risk of giving birth to a preterm baby.”

Even though cervical cancer in this country is largely under control, women still get it, and when they do, it can be devastating. Thomas J. Rutherford, Ph.D., M.D., FW ’94, associate professor of obstetrics, gynecology and reproductive sciences and director of gynecological oncology, recalls a patient in her mid-30s who was pregnant. The results of a routine Pap smear were abnormal. A colposcopy revealed a very high-grade squamous cell lesion. To save his patient’s life, Rutherford recommended an immediate radical hysterectomy, but that would have meant losing the baby. “The patient finally agreed,” Rutherford says, “but after the surgery she said to me, ‘I can’t believe I gave up one of my children.’ It was a difficult choice she made, but she probably would have died if she hadn’t.”

Another patient was a 20-year-old college student who had adenocarcinoma of the cervix, which is also caused by HPV. She underwent a cone biopsy, but the Pap smear still revealed abnormalities in her cervical cells, “We couldn’t repeat the procedure, because she wanted to have children,” Rutherford recalls. “We put everything on the table: This is the situation. Your best option is to have a child now.” The patient took Rutherford’s advice and had a baby, after which Rutherford performed a radical hysterectomy. “There she was, getting married, having a baby and then having a hysterectomy, all before she turned 21,” he says. “I assure you that wasn’t what she foresaw for herself.”

Even when a patient isn’t diagnosed with a precancerous lesion, the ordeal of getting a positive test result, going back for more tests and possibly having to have a colposcopy or a biopsy before finally getting a clean bill of health is stressful. “It’s also a very expensive way to prevent cervical cancer,” Brandsma says. “It’s a lot of money and anxiety.”

A far better approach, she and other HPV experts say, would be to vaccinate people against the disease. Researchers at Yale and elsewhere have been working on two types of vaccines with promising results. A prophylactic vaccine being developed at the National Cancer Institute and the University of Washington, among other places, would prevent infection by generating a neutralizing antibody. Brandsma, DiMaio and other researchers at Yale and elsewhere are working on a therapeutic vaccine that would generate killer T-cells that could recognize tumor cells as being foreign and destroy them. “Cervical cancer is the ideal cancer in which to demonstrate the principle of anticancer vaccines, because we know what the tumor antigens are. Viral E6 and E7 are the oncoproteins expressed in all lesions. They’re always required,” Brandsma says.

Two versions of the prophylactic vaccine have shown encouraging results in clinical trials. Both prevented persistent infection by the HPV types contained in the vaccine in 100 percent of vaccinated women and reduced cervical abnormalities by more than 90 percent. Merck & Co., the maker of one vaccine, reported in the fall of 2005 that, in a Phase III trial of more than 12,000 women, the vaccine prevented virtually 100 percent of growths that can lead to cervical cancer. The company has since announced plans to file for approval with the U.S. Food and Drug Administration before the end of the year. GlaxoSmithKline, maker of the other vaccine, reported similarly positive results with its clinical trials and plans to seek approval in Europe and other countries in 2006.

Once a vaccine is in widespread use, experts predict an immediate 44 to 70 percent reduction in abnormal Pap smears and a long-term reduction of close to 95 percent in cervical cancer rates. As promising as these numbers are, the vaccine also has limitations, chief among them being that three injections are required and the vaccine must be kept refrigerated. Especially in developing countries, where the need for a vaccine is the greatest, these obstacles have the potential to limit the vaccine’s efficacy. Another limitation is that, although the vaccine prevents infection by the most common high-risk HPV types, less common high-risk HPV types are not included.

Beyond that, the vaccine raises thorny social issues. To maximize its effectiveness, it should be given to girls between the ages of 9 and 12, before they become sexually active. Already, some religious groups have raised concerns that this will be interpreted as a license to engage in premarital sex.

“These vaccines could provide a huge public health benefit,” Lockwood says. “To allow their introduction to be blocked because of some extreme ideological position is unconscionable and irrational. It would be a huge cost savings, and could save some young person from dying in her 20s or 30s.”

Putting cancer genes to sleep
Vaccines are not the only approach to controlling cancers with viral origins. Using the HeLa cell line, which contains HPV DNA, researchers have figured out that the proliferation of cervical cancer cells requires the expression of the HPV oncogenes E6 and E7, which are expressed by cervical carcinoma cells. These oncogenes inactivate the cancerous cells’ major tumor suppressor pathways, thereby allowing the cells to proliferate.

An effective way to combat this, scientists have learned, is to induce a biological phenomenon known as senescence, an irreversible suspended animation of the cell, which acts as an important tumor suppressor mechanism. DiMaio and his colleagues have determined that the introduction of the papillomavirus protein E2 to the cell represses E6 and E7, halts cell growth and induces senescence. So, although the tumor cells have accumulated essential mutations, they still depend on the viral proteins. DiMaio likens it to a house of cards. “You need many cards to build a multistory house, but the whole edifice tumbles down if you remove the crucial card at the bottom.

“When we added E2, it induced senescence in a day or two,” DiMaio says. “This creates an important barrier to tumor formation and growth. It also gives us a new model to study senescence.” DiMaio says this is important because the hope is that senescence can be applied to other cancers as well. Also, there’s great interest in someday inducing senescence to block aging and age-related disease. “Half of my lab is focusing on senescence,” he says.

As the study of tumor virology continues to grow in importance and application, a growing number of Yale researchers are investigating other pieces of the puzzle. John K. Rose, Ph.D., professor of pathology, is interested in vaccines constructed from virus vectors. He is collaborating with Brandsma’s and DiMaio’s labs to develop HPV vaccines using a slightly different approach. The same antigen is involved, but instead of injecting DNA into the animal, as Brandsma does, he uses virus vectors. Rose is also in charge of a small unit that has recently recruited two junior tumor virologists. Michael D. Robek, Ph.D., assistant professor of pathology, studies replication of hepatitis B virus, and Robert E. Means, Ph.D., assistant professor of pathology, studies ways that herpes viruses avoid the immune response.

More than 50 years ago, Henrietta Lacks was helpless against the cancer that destroyed her body, but today, thanks in part to her cells, researchers are closer than ever to defeating that enemy, and the hope is that with the knowledge gained by studying HPV, other cancer-fighting breakthroughs will soon follow. YM

Jennifer Kaylin is a contributing editor of Yale Medicine.