Wednesday, December 17, 2014

How ex-Penn scientist's hunch led to cervical vaccine

Pediatrician Charles Scott had to speak louder and louder to be heard. Soon, he was almost yelling at 14-year-old Brianna Furrow as he counseled her about Gardasil, the world's first cervical cancer vaccine.

Brianna's mother, Patricia Furrow, scowled at her sons, ages 7 and 5, who were boisterously sharing Nickelodeon magazine in a corner of the small Medford examining room. "I need you to shut it," she hissed at them.

For Scott, accustomed to inoculating the diapered set, it was strange to be talking, much less yelling, about gynecological cancer and human papillomavirus. HPV is the sexually transmitted germ that is the target of Merck & Co.'s cancer vaccine, approved in June for females ages 9 to 26.

Only Brianna seemed at ease. True, the Cherry Hill ninth grader blushed at an oblique allusion to her virginity. But on this September afternoon, she was ready to join this revolutionary war on cancer. To be part of the first generation to combat cervical cancer simply by rolling up its sleeves. She had good reason.

Her neighbor has cervical cancer, Brianna explained, and she'd been telling girls to get vaccinated.

Four thousand miles away, at the German Cancer Research Center in Heidelberg, the man whose out-of-the-box thinking in Philadelphia paved the way for the vaccine was asked to reflect on the triumph. Did he feel prescient or vindicated, maybe with a twinge of bitterness?

"I'm pleased," said Harald zur Hausen, 70, with his usual understatement. "It's gratifying."

Forty years ago, during a postdoctoral fellowship at the University of Pennsylvania, zur Hausen suspected that the same virus that caused ordinary warts also seeded cervical cancer.

His hunch was considered outlandish. Not only were researchers closing in on a much likelier viral culprit, but everyone knew warts were harmless. The ubiquitous, cosmetically annoying papillomavirus could not possibly cause half a million cervical cancer diagnoses and 250,000 deaths globally each year.

And yet zur Hausen was right. His lab showed that HPV was a family of viruses, and that the two most dangerous types - both part of the new vaccine - were involved not only in cervical cancer, but also in rarer malignancies. Men and children, as well as women, were vulnerable.

In the end, zur Hausen would also become a prophet of a scarier idea now recognized as truth: Certain viral infections can culminate in cancer. HPV is just one.

For more than a decade, zur Hausen slogged against skepticism and the limits of technology, confident that the ultimate reward would be a cervical cancer vaccine, and a chance for a public-health victory on par with vanquishing polio.

It's surprising, then, that other scientists wound up with that reward. Scientists who turned to zur Hausen for virus samples developed the vaccine technology; their institutions won the patents, the deals with drug companies, the credit.

What happened?

Sitting in his office, the genteel virologist sipped his morning coffee, sprinkled with cocoa. His thinning white hair set off his eyes, blue as robin's eggs.

It was what didn't happen that mattered, he said.

A lucky break

The youngest of four, zur Hausen was just a boy when he started wondering about the origins of diseases, but he insists he wasn't studious.

In German secondary school - which starts in fifth grade - he was "struggling for the first one or two years. My first report card said, 'Upgrading to the next grade is possible, but it occurs with serious concern. '"

This is self-deprecating, considering he never attended second or third grade. World War II bombing raids shut down the schools in his town.

Still, it was mostly luck - and a bit of trash - that led him to Philadelphia in 1965.

He was 29, a physician, and had spent four frustrating years doing random research at the Institute of Microbiology in Düsseldorf. He wanted more guidance and training in molecular technology, which meant he would have to leave, maybe go abroad.

Coincidentally, at Penn, virology professors Werner and Gertrude Henle were seeking a bright young German to work in their lab at Children's Hospital of Philadelphia. The married couple were themselves expatriates - Werner, whose grandfather was a Jew, had fled the Nazis - so they wrote to the director of the Institute of Microbiology, who passed the letter to a department head, who couldn't think of anybody to recommend.

He threw the paper away.

Hours later, the department head ran into zur Hausen, and it dawned on him to mention the missive.

"I raced down the hall, pulled the letter out of the trash can, and very quickly wrote to Werner," zur Hausen said.

The rest would be history. Years later, in correspondence now archived in the U.S. National Library of Medicine, he told the Henles what joining them meant:

"I find it the most important decision I made in my scientific life, although I certainly was not aware of it at the time."

The Philadelphia story

Zur Hausen arrived in Philadelphia at the end of 1965, settling into a cozy Drexel Hill apartment with his wife and the first of their three sons.

It wasn't long before his new bosses realized they had "a winner," as they later described him, a maverick thinker who bucked conventional wisdom - including their own.

The Henles were already known for their work on mumps and flu vaccines. Now they were searching for viruses that existed only in theory - ones that caused human cancer.

They had just published a paper showing an immune response to the recently discovered Epstein-Barr virus in patients with Burkitt's lymphoma. The dreadful disease, which caused a disfiguring jaw tumor, was the leading killer of children in equatorial Africa.

While this evidence of a link between the virus and the cancer was exciting, it was circumstantial. To bolster the link, the Henles tried to find the virus in thawed tumor biopsies.

That was when things got puzzling.

They knew that a virus cannot reproduce on its own. It sneaks into a cell and, like a parasite, hijacks the host's molecular machinery to copy itself. Many times over. Then the "virus particles" emerge from the cell and invade others.

But looking through an electron microscope, the Henles couldn't find Epstein-Barr virus particles in the thawed tumor cells, no matter how they processed or stained the sample. Only after they grew the lymphoma cells in a lab-dish "culture" were virus particles suddenly visible. That meant the virus was in the thawed cells - somewhere - to begin with.

How could the virus be in cells that it was not using to make infectious particles?

Zur Hausen had a hunch: Maybe Epstein-Barr shed its outer shell and slipped its genetic material into the host cell's genome. This could explain how the virus was overlooked by the immune system, as well as how it promoted malignancy - by mucking up the host's DNA.

No one had ever demonstrated such a thing in human cells. Werner and Gertrude were skeptical. But with Werner's blessing, zur Hausen devoted his three years in Philadelphia to pursuing evidence. In 1970, he published his work in the influential journal Nature.

His understanding of "viral DNA integration and persistence" would shape his insights into the papillomavirus.

And he would stick with his hunches.

A crazy idea

For centuries, physicians had noticed a connection between sex and cervical cancer.

In 1842 in Italy, a doctor observed that prostitutes and married women died of cervical cancer, while nuns didn't - although he blamed "smegma," dirt under the foreskin of the penis.

With more sophisticated understanding of germs, the blame shifted. Maybe it was a sexually transmitted bacteria like syphilis. Or maybe even a virus.

By the 1960s, the leading candidate was herpes simplex-2, the new cringe factor in dating because so many people were suffering from the virus' painful, oozing genital sores.

Circumstantial evidence linked cervical abnormalities and herpes simplex - for example, women with one often had the other - but no one could find the virus in cancer cells. Indeed, herpes got attention partly because it was so easy to grow and study in culture.

Zur Hausen doubted the herpes hypothesis. Then, one day in the Henles' lab, he skimmed a 1967 article titled "Human Wart Virus" in a back issue of Bacteriological Reviews.

All human warts - on the skin, the soles of the feet, the genitals, and, rarest of all, the larynx or vocal cords - were believed to be caused by a single type of papillomavirus. The paper's authors concluded that while the virus seemed innocuous, unusual examples of ominous cell changes in animals and humans raised "the possibility that it may at times be responsible for less benign tumors."

They were not the first to think so. In some animals species, cancer viruses had even been shown to exist.

But while herpes simplex was ideal for lab studies, the human wart virus was infuriating. It wouldn't grow in culture, and it wouldn't grow in test animals. So it had been mostly ignored.

Zur Hausen was intrigued. He casually searched old medical journals to see whether the wart virus might be involved in cervical cancer. Sure enough, he found a clue: Several anecdotal cases of genital warts that turned malignant.

He returned to Germany and continued his groundbreaking research with the Henles, linking Epstein-Barr to a rare nasalpharyngeal cancer.

But he couldn't stop thinking about the wart virus, and his hunch.

In 1974, zur Hausen, then 38, traveled to a cervical cancer convention in Key Biscayne, Fla. He listened to experts present the "mounting" evidence that herpes simplex was the bad actor in cervical cancer.

Then he took the lectern and respectfully said: I think it's the wart virus.

There was hostile silence. While the herpes evidence might be inconclusive, zur Hausen's was perceived as nonexistent. Basically, all he had was 100 rare, anecdotal reports from the medical literature of cancerous genital warts.

"It was still a crime," he wrote to Werner, "to raise doubts."

In full swing

While Zur Hausen turned his attention to cervical cancer, the sexual revolution was in full swing.

Young American women routinely went on "the pill" and slept with their boyfriends.

They didn't realize that the cervix - an inch-long tunnel between the vagina and the womb - has an area where immature and mature cells overlap, forming the dynamic microenvironment that HPV needs to flourish (and can't get in culture). Or that because of their youth, this "transformation zone" was particularly large and vulnerable.

Soon, an unprecedented number of them were getting bad news from their doctors: abnormal pap smears.

U.S. cervical cancer rates had been dropping since Cornell University pathologist George Papanicolaou's eponymous test began to catch on in the late 1950s. It capitalizes on the fact that cervical cancer is one of the few killers with well-defined, slow-progressing precancerous stages. A scraping of cervical cells under a microscope could reveal a problem in time for tissue to be excised, thus preventing a potential cancer a few decades down the line.

The test often missed abnormalities, but that was OK. Most mild lesions got better without treatment. Indeed, though no one knew it at the time, HPV infections often came and went harmlessly. With regular screening, a woman's lifetime risk of cervical cancer was less than one percent.

Katie Allen, 59, of Anaheim, Calif., was not very regular. Like most women, she hated lying on a table, feet in stirrups, vagina pried open with a metal speculum.

"Which was stupid," the retired archaeologist says now.

In 1995, she noticed blood after she and her husband made love. As the intermittent bleeding grew worse, she blamed her ebbing estrogen.

But it was not menopause. Surgery revealed she had cervical cancer that had spread to her pelvic lymph nodes.

After 30 treatments of external pelvic radiation, she spent two 24-hour periods in a lead-lined room with radioactive material sewn inside her. Even so, her chance of survival was about 60 percent.

She remembers the gynecological oncologist saying, "Not to worry. You still have the option of a pelvic exenteration."

An exenteration is close to an evisceration. The bladder, rectum, and part of the large intestine are removed. Thankfully, it was unnecessary.

Allen is not surprised that epidemiologists have linked an upward trend in cervical cancer among women of her generation to the sexual revolution of their youths.

Of course, in countries where health care is a luxury, there has never been a downward trend. Globally, cervical cancer is the second most common cancer in women, killing 250,000 a year, usually in the prime of life.

Tedium and trip-ups

In the late 1970s, young scientists at the University of Freiburg were eager to join the chairman of the virology department in his quest for the cause of cervical cancer.

Zur Hausen assumed that isolating and mapping the DNA from a genital wart would be a key breakthrough. Presumably, this DNA could be used as a molecular probe to find its evil twin in cervical cancer cells.

But since the virus wouldn't grow in culture, his students had to force it out of fresh genital warts for each experiment. A parade of doctors delivered the ugly protuberances, which were then minced, pulverized, dissolved, and spun in a centrifuge.

This rarely yielded virus particles, but even when it did, zur Hausen's team couldn't tease out the prize - pure viral DNA.

The researchers were learning that tedium and trip-ups outnumber triumphs, and progress tends to creep, not leap, ahead.

"It was hard to keep the postdocs interested," zur Hausen recalled. "Not everyone was convinced this was a good project."

They read enviously about more promising projects to find genes that could turn cancer on, and wished they, too, were researching "oncogenes."

But they kept creeping.

French virologist Gerard Orth narrowly beat zur Hausen to publishing the world's first DNA map of a wart virus, isolated from plantar warts. Zur Hausen's group came back with the maps of other skin wart viruses, and declared that "the" wart virus was actually a varied family. The world's wart virus experts - all 16 of them - agreed to number the HPVs in order of discovery.

In 1980, zur Hausen's lab finally got a virus out of genital warts, HPV-6. But it was a dead end. His assumption that it would lead to the cancer virus was wrong.

But his overall hunch that the virus caused cancer was right. He was sure of it.

A new thought occurred to him. Maybe there was a spectrum of HPVs, ranging from those with a negligible risk of cancer, such as the genital wart version, all the way to high-risk strains.

They would have to look harder.

Sometimes harmless, sometimes deadly

The most dramatic example of the enigmatic interplay of HPV, its hosts and the environment is a disease few people have heard of: recurrent respiratory papillomatosis.

Kristina Wilson, 32, of Nashville, had never heard of it. All she knew was that her second child, Morgan, was born with an unusually faint cry.

The pediatrician said, Be glad she's not a screamer. But at 9 months, Morgan almost suffocated. Doctors discovered vocal-cord warts, and threaded an instrument down her throat to eradicate them.

Within two months, they were back.

"She's had about 77 surgeries," Wilson said of her daughter, now 9.

In children, the infection is presumably transmitted at birth, when a child's throat comes in contact with a mother's genital warts - a presumption that periodically sends Wilson into a guilt-ridden depression.

Yet for unknown reasons, only seven out of every 1,000 children born to mothers with genital warts develop vocal-cord warts. There are even cases of twins in which only one has the disorder.

Laryngeal warts are just as mysteriously rare in adults - an estimated 3,600 new cases a year - although the presumed transmission mode, oral sex, is common.

Respiratory warts can go away after a few bouts of hoarseness, or stubbornly grow back. Or, in rare cases involving high-risk HPVs, progress to cancer.

Kathy Blankenship, 51, of Hillsboro, Ore., was diagnosed in 2000 after six years of voice problems. After treatment that included steroids and radiation - which she belatedly learned can promote malignant transformation - her laryngeal warts turned to cancer that spread to her lungs.

She now belongs to an online support group. "But I want to make sure I don't scare them," she said in the robotic machine voice that has replaced her own. "Obviously, my case has been unusually horrific."

The breakthrough

Matthias Duerst, a 1981 addition to zur Hausen's lab, was excited. Using the lab's latest HPV - isolated from laryngeal warts - he found a fragment of viral DNA in a cervical cancer biopsy.

He showed the data to his busy boss, who was soon to become the chair of the German Cancer Research Center. Zur Hausen reacted unemotionally, as usual.

See if you can clone it, he directed. Cloning was a relatively new technology that could fill in missing parts of a DNA sequence, then make a vast number of complete copies.

In 1983, Duerst succeeded.

Cloning this virus - HPV-16 - was the true breakthrough. Cloning provided a fuller picture, a complete genetic sequence that they could use to look for the viral DNA in the biopsies, instead of merely fishing for fragments.

They found HPV-16 in more than half of cervical cancer biopsies. They found it in severely abnormal cervical lesions. They found it in some vulvar, anal, penile and neck cancers.

Even zur Hausen was ecstatic.

The pace of discovery quickened. The next year, the lab cloned another "high-risk" strain, HPV-18.

It wasn't proof of causation, but it was the smoking gun.

The team zeroed in on the event that pushed cells beyond abnormality to cancer. HPV-16 or 18 randomly inserted two "oncogenes" into the cell's genome, where they ducked the immune system, genetically destabilizing the cell.

Echoes of the Epstein-Barr virus.

Rather than getting patents to restrict access to the viral clones, zur Hausen distributed them to researchers around the world.

They went on to decipher the mechanisms of carcinogenesis: One viral oncogene tricked the cell into multiplying like mad - creating more cells for the virus to hijack, and more chances for the dividing cell to accidentally introduce mistakes in its own genome. The other oncogene kept the cells from dying.

Uncontrollable, immortal cells. The definition of cancer.

After a decade of scorn, zur Hausen was an instant genius. His calendar filled with invitations to lecture all over the world. He had found the viral link.

He was vindicated.

What didn't happen

What happened next was not what he expected.

By 1984, he was confident that a vaccine was within reach. He approached five Swiss and German pharmaceutical giants about funding research. Traditional vaccine technology - using cell cultures and inactivated viruses - would not work, but he had some ideas for alternative approaches.

Zur Hausen thought he had made a good case. The molecular evidence was overwhelming. He had clout and credibility.

Yet only one company, the former Behringwerke, gave him some seed money.

Then promptly backed out.

The problem was that a link was still missing. Big epidemiological studies were needed to show the prevalence and pattern of disease in actual populations of women. As such studies began to be published, the results were often bizarre; some found that 100 percent of women were carrying the high-risk strains even though they had normal Pap smears.

Eventually, it became clear that the new ultrasensitive DNA detection methods were prone to error in inexperienced hands. But as late as 1989, the World Health Organization's cancer research arm still doubted the link between HPV and cancer, calling the evidence "suggestive."

This, then, was the thing that could have happened but didn't - the missed opportunity that still stings zur Hausen. Not just because other labs sprinted past his, but because women's lives were at stake.

It would take 22 more years before girls like Brianna would be getting a vaccine in their doctor's office.

In a 2001 editorial in the Journal of the National Cancer Institute, zur Hausen lamented the lost time.

"In the personal experience of this author, up to this period it has not been possible to convince the pharmaceutical industry to consider the initiation of vaccination programs against high-risk HPV infections. In all likelihood, several years have been lost. If we calculate . . . 400,000 cases per year globally, this delay may turn out to be costly. "

Could a vaccine have come sooner?

Doubtful, say some of the inventors.

Incremental advances in the early 1990s by at least five groups in the United States and Australia led to the vaccine technology. Basically, they figured out how to make a hollow, empty human papillomavirus shell that primes the immune system to recognize and attack the real thing.

Gardasil, the first of numerous versions in the pipeline, wards off the primary carcinogenic types (HPV-16 and 18) and the genital-wart types (HPV-6 and 11).

"But to tell you the truth, people wouldn't have started working on these unless Harald had found HPV in cervical cancer," said pathologist Richard Schlegel at Georgetown University, which holds the dominant U.S patent.

"Zur Hausen's work was seminal," echoed microbiologist Richard Rose at the University of Rochester.

The Rosetta stone

Zur Hausen usually eats at his desk in the tumor virology building, erected during his 20-year tenure as head of the German Cancer Research Center. But on a day in October, he took time for lunch at the tennis club near campus.

At 70, he is busier than ever, editing the International Journal of Cancer, working on a nasal-spray form of the HPV vaccine, and lecturing around the world on cancer viruses. Moscow, Lima, Bangkok and London are in the coming weeks.

He could rightfully claim - if it were his style - that he is not only vindicated, but visionary. Twenty percent of cancers are now estimated to be caused by viruses, either directly or indirectly. These include Epstein-Barr, the hepatitis B and C viruses, and the AIDS virus.

"Viruses have been key instruments in the revolution in cancer biology that has occurred over the last 20 years," Baylor College of Medicine virologist Janet S. Butel wrote in the journal Carcinogenesis. "Viruses have turned out to be the Rosetta stone for unlocking the mysteries of cell-growth control."

Finishing off lunch with a cup of coffee, he removed his glasses. Yes, his HPV work led the field forward. But there are many more cancer viruses to be found.

"This is not the end of the story," he says quietly.

He's just sure of it.

Marie McCullough INQUIRER STAFF WRITER