# News this Week

Science  28 Mar 2014:
Vol. 343, Issue 6178, pp. 1412
1. # Around the World

1 - Yunnan province, China
New Virus Suspected in Chinese Deaths
2 - Almería, Spain
3 - Southeastern Guinea
Ebola Outbreak Kills Dozens
4 - London
U.K. Budget Prioritizes Big Data, Cell Therapies, and Graphene
5 - Brussels
World Map of Environmental Conflicts Launched

## Yunnan province, China

### New Virus Suspected in Chinese Deaths

Research into the deaths of three men in southwestern China has turned up an intriguing new viral species. The men contracted severe pneumonia and died in June 2012 after removing slag from a derelict copper mine. Analysis of anal swabs from rats in the mine revealed a potential killer: a virus resembling those in the Henipavirus genus, scientists reported in the June issue of Emerging Infectious Diseases.

Two of the three confirmed henipavirus strains are deadly to humans, and all three appear to circulate in the wild in fruit-eating bats called flying foxes. Bats and shrews in the mine in Yunnan tested negative for the new virus, named Mojiang paramyxovirus (MojV); only rats were infected. MojV "could be a 'bridging' virus between those in bats and rodents," says Lin-Fa Wang of the Australian Animal Health Laboratory in Geelong. A survey of 38 bat species across China prompted by the MojV find failed to turn up any henipaviruses. http://scim.ag/_henipa

## Almería, Spain

A tight budget plan imposed by the funders of the German-Spanish Astronomical Center at Calar Alto (CAHA) has driven the center's director to resign. José María Quintana plans to step down in late April because the Spanish National Research Council and Germany's Max Planck Society, which jointly operate the observatory, support a funding strategy he considers too harsh.

An operating plan signed in May 2013 set aside €1.6 million per year until 2018 for CAHA, whose Potsdam Multi-Aperture Spectrophotometer has been used to survey nearby galaxies. When Quintana assumed the post in June 2013, he advocated budget increases that he says would have kept the center's three telescopes running and retained core staff. But he announced his resignation in late January when the funders opted to keep post-2014 funding flat. Spanish media reports say that since then, CAHA's interim managers have fired cooking and cleaning staff and restricted the operation of one telescope by 10 days a month. While still hopeful for the observatory's future, Quintana foresees an era of "unreasonable minimums." http://scim.ag/_CAHA

## Southeastern Guinea

### Ebola Outbreak Kills Dozens

The first large human outbreak of Ebola hemorrhagic fever in West Africa has killed dozens of people in Guinea and may have moved into neighboring Liberia and Sierra Leone. On 25 March, the World Health Organization said that 86 cases with 59 deaths had been documented in four districts in southeastern Guinea, and at least six suspect cases, with five fatalities, were being investigated across the border in Liberia. The culprit is the Zaire ebolavirus (ZEBOV), says virologist Sylvain Baize of the Institut Pasteur in Lyon, France, who with his colleagues analyzed initial samples from the outbreak. ZEBOV is the deadliest of the five known Ebola species.

This is the first outbreak of ZEBOV outside Central Africa, and Baize says it will be important to try to understand how the outbreak started. Fruit bats have been found to be asymptomatic carriers of the virus, and they are strongly suspected as a reservoir where Ebola hides between outbreaks, but, Baize notes, the field studies to find a definitive culprit "are quite difficult."

## London

In a 2014 budget statement, U.K. Chancellor of the Exchequer George Osborne announced £222 million ($366 million) in new funding for research projects. Forty-two million pounds will go to create the Alan Turing Institute for Data Science, which will focus on the analysis and application of big data. Another £74 million will help set up a center to manufacture cell therapies for clinical trials and a lab to research new technologies using graphene. And £106 million will go toward 20 new centers for doctoral training, Osborne announced on 19 March. While researchers have largely welcomed the new funding, the Campaign for Science and Engineering (CaSE) points out that the core science budget, which has remained stagnant since 2010, devotes only 1.72% of Britain's gross domestic product to research and development—significantly less than many other developed nations including Germany, the United States, and Japan (all around 3%), and the European Union average of 2.08%. The flat budget "is biting scientists and engineers and squeezing universities," and may deter outside investment, CaSE Director Sarah Main said in a statement. ## Brussels ### World Map of Environmental Conflicts Launched Want to see which communities around the world are fighting off a project, such as a mine or a dam, that could harm the environment? A new online "atlas of environmental justice," developed by a group of academics and activists funded by the European Commission, allows just that. Activists, scholars, or the purely curious can search and filter the data across 100 fields, including by commodity, company, country, and type of conflict. The atlas, launched last week, was built by Environmental Justice Organisations, Liabilities and Trade (EJOLT), an E.U.-funded project that brings together 23 teams from academic and activist organizations. It now lists 915 conflicts; 271 involve local scientists and professionals, and 17% are described as environmental victories. "It's only the tip of the iceberg," says EJOLT spokesman Nick Meynen. The organization aims to raise the coverage to 2000 conflicts in the coming year, which would enable quantitative analyses. Academics can provide input to expand the range of cases and fill the gaps—including blank spots in China. http://ejatlas.org/ 2. # Random Sample ## Noted The World Health Organization (WHO) was set to declare India and the 11-country Southeast Asia region polio-free on 27 March. Long the global epicenter of the virus, India saw its last case on 13 January 2011. That leaves two more WHO regions to go: the Eastern Mediterranean Region, where Afghanistan and Pakistan are the roadblocks, and the African region, where Nigeria remains endemic and has sparked nearby outbreaks. ## Champions of the Sargasso It may lie beyond the jurisdiction of any nation, but the Sargasso Sea—and the hundreds of marine species it supports—has many advocates. Last week, a team of 33 researchers with the Danish Eel Expedition 2014 arrived in this open-ocean region of the North Atlantic to study the declining European eel populations that spawn there. Using fine-mesh nets (pictured above) to catch larvae, they hope to "plug the main gaps in our knowledge" of the eel's early diet and breeding habits, says the project's leader, Peter Munk of the National Institute of Aquatic Resources at the Technical University of Denmark in Charlottenlund. The team also will examine whether climate-related changes to ocean currents are contributing to the eel's decline. Meanwhile, five countries, including the United States and the United Kingdom, signed a nonbinding agreement earlier this month to protect the area from overfishing and pollution. The organization behind the agreement, a partnership between scientists, conservation groups, and the government of Bermuda called the Sargasso Sea Alliance, released a film celebrating the agreement, available at https://vimeo.com/89868953. ## By the Numbers 7 million—Deaths worldwide in 2012 as a result of indoor and outdoor air pollution exposure, which amounts to one in eight total deaths, according to a new World Health Organization estimate. ## DNA's Forgotten Discoverer James Watson and Francis Crick may be the names most associated with DNA, but a German biotech company is determined that the world never forgets Friedrich Miescher, the Swiss scientist who first discovered the molecule, nearly a century before the pair famously deduced its structure. CureVac, a company based in Tübingen, Germany, that develops RNA-based vaccines and therapies, won a €2 million prize for innovation from the European Union this month and announced plans to use some of that money to restore the University of Tübingen lab where Miescher made his discovery. Together with the university, the firm wants to transform the lab, the former kitchen of the old town's medieval castle, into a public exhibition about his work and legacy. (The university now uses the space as a computer room.) While studying the composition of pus cells in 1869, Miescher found a molecule in cell nuclei that was not a protein, but a substance "not comparable to any hitherto known group." He intuited that the molecule, which he dubbed "nuclein," played a role in heredity, but didn't believe that a single molecule could lead to a huge variety of individuals and species, says Ralf Dahm, of the Institute of Molecular Biology in Mainz, Germany, who has written about Miescher's work. He says Miescher has kept a low historical profile, in part due to his "introverted" and "insecure" personality. "He's one of the most important guys in science," says CureVac CEO Ingmar Hoerr, "and not many people are aware of it." 3. # Newsmakers ## Tyler Prize Goes to Ecologist With a Mathematical Eye Whether he's looking at a colony of mussels or the international balance of political power, Princeton University ecologist Simon Levin sees the same thing: complex systems. This week, Levin was named the 2014 recipient of the$200,000 Tyler Prize for Environmental Achievement for a 4-decade career that connects mathematical concepts such as game theory to the dynamics of animals and ecosystems.

Levin's work has influenced Pacific salmon recovery efforts and protection of the Hudson River, but it has also found some unlikely applications in topics beyond environmental science, from the destructive dynamics of cancer cells to global affairs in the age of terrorism.

Levin says he's encouraged that a complex, interconnected view of ecosystems is triumphing over more "simplistic" management schemes. "What you're really trying to preserve are the broad, emergent features of the system," he says, "whether it's a forest system or a financial system."

## Biomedical Donors Leave Research Legacy

Two of science's most generous philanthropists recently passed away. On 19 March, Patrick McGovern, who became one of American's wealthiest people after founding the publisher International Data Group, died. McGovern received an undergraduate biophysics degree at the Massachusetts Institute of Technology in 1959 and 4 decades later, he and his wife donated $350 million to create a neuroscience research institute at his alma mater. James Stowers, who died on 17 March, originally studied medicine before moving into business and making his fortune running an investment firm. In 1994, after both had run-ins with cancer, he and his wife established the independent Stowers Institute for Medical Research in Kansas City, backing its endowment with more$2 billion in assets. The institute opened in 2000 and now has nearly 400 scientific staff members.

4. # The Camel Connection

1. Kai Kupferschmidt

Two years after the first recorded deaths from MERS, most scientists agree that camels are key to the spread of the deadly disease. But containing it won't be easy.

On 21 March 2012, a 25-year-old student in Jordan started coughing. A few days later, he developed fever and shortness of breath. He was admitted to a hospital, where he ended up in intensive care. By the time he died on 25 April, several nurses and doctors had developed similar symptoms. One of them died, too. Then the mystery disease disappeared again.

Two years later, it's clear that the outbreak was the prelude to a protracted struggle against what is now known as the Middle East respiratory syndrome (MERS) virus. And although scientists still have lots of unanswered questions, evidence is mounting that camels play a key role in spreading the new pathogen. The camel-borne threat may extend far beyond the Middle East. Last month, a team led by Malik Peiris, an infectious disease researcher at the University of Hong Kong, showed that almost all camels in four Egyptian abattoirs had MERS antibodies in their blood; most had been imported from Ethiopia and Sudan, suggesting the virus may reside in large parts of Africa.

As a result, scientists are shifting their focus from human cases to camels. "It is becoming ever clearer that MERS is a classic zoonosis," says virologist Christian Drosten of the University of Bonn in Germany. "We need to concentrate more on the animals." One idea gaining traction is to vaccinate camels. "Protecting camels right now may be the single most important thing we can do to protect humans," says Michael Osterholm, director of the Center for Infectious Disease Research and Policy at the University of Minnesota, Twin Cities.

But vaccines against coronaviruses, the group to which MERS belongs, are difficult to make, and researchers face hurdles ranging from money problems to the lack of a suitable animal model. Neither veterinarians nor health officials in the Middle East have risen to the challenge. To make matters worse, there are very few labs capable of safely studying deadly viruses in animals as big and unruly as camels.

## The evidence mounts

After it was identified in June 2012, MERS quickly made its way to the top of the global public health agenda. The virus is related to severe acute respiratory syndrome (SARS), a deadly pathogen that emerged in China in late 2002, killed 800 people around the world, and nearly caused a pandemic. The World Health Organization (WHO) still worries that MERS could be the sequel to that frightening episode. But while the disease has sickened at least 199 people and killed 84, it has largely been confined to the Middle East (see map, p. 1423).

The first clue that camels might be involved came from a patient from Abu Dhabi who died in a German hospital in March 2013. He owned racing camels and reported having had close contact with a sick camel before falling ill. Then in August, a team including Marion Koopmans, now at Erasmus MC in Rotterdam, the Netherlands, reported that 50 retired racing camels from Oman all had antibodies against MERS in their blood, which is evidence of a past or current infection. None of the cows, goats, and sheep they tested had such antibodies.

Some scientists were skeptical. Other coronaviruses known to infect domestic animals could have caused the positive tests, they argued. "We don't think camels have anything to do with it," said Ziad Memish, an infectious disease specialist and Saudi Arabia's deputy health minister, at the time. "It just seemed so weird," says Peter Daszak, a veterinary epidemiologist at the EcoHealth Alliance in New York City. Genetically, "the virus looks like a bat virus." But later, MERS antibodies were reported in camels in Jordan, Egypt, the United Arab Emirates, and Saudi Arabia. And scientists investigating an outbreak on a farm in Qatar found snippets of the genome of the MERS virus in nasal swabs from three camels, suggesting that these animals were actually infected at the time.

Last month, a team led by Columbia University virologist Ian Lipkin reported evidence of a MERS infection in three-quarters of more than 200 Saudi Arabian camels tested; they also recovered MERS viral sequences that proved an exact match to viruses found in humans. Because the researchers rarely found the virus in camel feces, Lipkin believes fine droplets from the animals' noses transmit it. Some of the positive samples dated back to 1992, suggesting that isolated, unrecognized human MERS cases may have happened for decades, he says. And just last week, scientists from Saudi Arabia and Europe reported about a MERS patient who fell ill after caring for sick camels; they managed to sequence about 15% of the viral genome from one of the camels, and that part was almost identical to the sequence of the human virus.

No one is suggesting that contact with camels is the only way humans can become infected. Infected people can also spread MERS, as clusters of infection—such as the one in Jordan—indicate. Memish now accepts that camels seem to transmit MERS, but he says that some so-called index cases—patients who did not contract the disease from another patient—say they had no contact with the animals. It's unclear how they got infected.

Some may have been exposed indirectly, says Anthony Mounts, WHO's point person on MERS. In one recent instance, a man who worked with camels remained healthy, while his wife developed MERS. "The husband could have brought the virus home on his clothing," Mounts says, though he adds, "this is not proven at all." Contaminated food could be another route. But to identify all possible routes, local knowledge is crucial, Mounts says. He recently learned that drinking camel urine and applying it to the skin is used as a natural remedy for a variety of ailments in the region. The practice is probably not the primary route of transmission, he says, but it's something that should be studied.

Other animals may still be involved. Bats seem to be MERS hosts, too; a fragment of viral RNA recovered from the feces of an Egyptian tomb bat living close to the first known Saudi MERS patient's house was identical to the patient's virus, so perhaps contact with bats can explain some cases. "It is important to remain open-minded about all potential sources of exposure for human and animal cases," a representative for the World Organisation for Animal Health writes in an e-mail.

If camels are MERS's main conduit, however, that puts the spotlight on countries far beyond the Middle East. Only about 260,000 of the world's 27 million camels live in Saudi Arabia. Africa has far more: 7 million camels in Somalia alone, 3 million in Kenya, and 1.5 million in Chad.

The virus probably circulates in these herds as well, Peiris says. His 27 February paper in Emerging Infectious Diseases presented some early evidence. The scientists tested 52 camel blood samples from four abattoirs around Egypt and found antibodies against MERS in 48 of them, mostly from camels imported from Sudan and Ethiopia. They also took nose swabs from 110 camels and found MERS RNA in four imported animals. It's very likely that infection has gone unnoticed in Africa, both in animals and humans, Peiris says. "Health authorities really need to test patients with severe pneumonia all across Africa for MERS."

For now, the MERS outbreak is generating a steady trickle of new cases: WHO reported four in January and four in February. But other zoonotic diseases have smoldered for years before they exploded, and no one knows whether MERS could also hold a nasty surprise. The fact that it didn't spread around the world after the Hajj, the massive pilgrimage to Mecca, in 2012 and 2013, has put some minds at ease. But Drosten says that may have been a lucky break. "Camels all give birth around the same time in the winter months," he says. They may contract MERS shortly after birth and be at their most infectious in spring. The Hajj is now in autumn, but because the Islamic year is shorter than the calendar year, it will eventually occur in the spring, Drosten says, "and that may change everything."

If camels are the problem, vaccinating them might be the solution. A camel vaccine would be far cheaper to make than a human one, because testing in animals is faster and easier. Keeping the animals healthy would also make economic sense because camels are valuable, Daszak says: "They're eaten; they're raced; there are beauty parades. The Saudi government is going to have to act on this." Breeding facilities would be the place to deploy a vaccine, says microbiologist Matthew Frieman of the University of Maryland School of Medicine in Baltimore. "We have an opportunity to vaccinate all newborn camels to block infection from older camels, which seems like it's what's happening," he says.

But it's not that simple. Camel breeders don't see MERS as a problem because it does not seem to make camels very sick, and perhaps not at all, says Juan Lubroth, chief veterinary officer of the Food and Agriculture Organization of the United Nations in Rome. Nor have veterinarians and animal health officials shown great interest. "I have not been very successful in engaging my peers at the national or regional level," Lubroth says. Most countries don't see MERS as an immediate threat, and the U.S. National Institutes of Health has not yet awarded any grants for vaccine studies. Microbiologist Shibo Jiang of the New York Blood Center in New York City says he could not stir up any interest among companies either, "because of the unpredictable market in the future."

Nor does anyone know how to make a MERS vaccine. Jiang believes the best target may be the virus's spike, a protein sitting on the surface that it uses to attach to cells. He and his colleagues fused a 212–amino acid piece of the spike to the stem of a human antibody. This vaccine caused mice to produce antibodies that protected cells in a petri dish from infection.

But coronavirus vaccines pose special challenges. One worrisome example is feline infectious peritonitis, a fatal cat disease. Cats carrying antibodies against this coronavirus are known to get sicker, instead of being protected. Fears that this might happen in humans, too, have plagued attempts to develop a SARS vaccine based on a whole, killed virus. The exact mechanism isn't quite clear, but Jiang found that with SARS, using just the part of the spike protein that binds to the cell receptor avoided the problem. He hopes the same will hold true for MERS.

But the receptor-binding area can change rapidly as the virus evolves, Frieman says, which might limit the protection offered by a vaccine. He has tried another approach: In collaboration with a company called Novavax, Frieman produced insect cells engineered to express the whole MERS spike protein. He harvests the protein as nanoparticles, small clumps of molecules sticking together. When these were injected into mice, the animals produced antibodies against MERS, Frieman reports in an as-yet unpublished paper.

A third strategy comes from Gerd Sutter of the Ludwig Maximilians University in Munich, Germany, who engineered a smallpox vaccine strain called MVA to carry the genetic information for the spike. The vaccine might kill two birds with one stone: It would act as a MERS vaccine in the animals but should also protect them against camelpox, making it more interesting to camel owners.

Testing vaccine candidates is the next hurdle. A good animal model for MERS has been hard to develop; the spike protein binds to a protein called DPP4 on the surface of human lung cells, but the murine version of DPP4 is so different that MERS does not infect mice. Rhesus macaques seem to become infected but show hardly any symptoms: "Without a CT scan it's hard to tell that these animals are ill," says Lisa Hensley of the National Institute of Allergy and Infectious Diseases' lab in Frederick, Maryland. Marmosets get sicker, but they are hard to come by. Other groups have tried mice, ferrets, hamsters, and guinea pigs; none of them worked very well.

So microbiologist Stanley Perlman of the University of Iowa has engineered an animal model: He packaged the human form of DPP4 into an adenovirus and then infected mice with it, coaxing them into expressing the protein on their cells' surfaces. The animals could then be infected with MERS, Perlman reported in a paper this month. How closely such a model mirrors what happens in camels or humans is unclear, however.

In fact, scientists still know disappointingly little about what MERS does in camels, says virologist Bart Haagmans of Erasmus MC. It's still unclear whether infected camels get sick, and at what stage they are most infectious to other animals or to humans. "There are a lot of questions that need to be answered in camel experiments," Peiris says. Haagmans says he would love to do those studies, but Erasmus MC doesn't have facilities to house the animals. Few labs around the world do.

Many other important questions about MERS remain unanswered, and scientists say that's in part because the affected countries have been uncooperative from the start. Are many people infected without showing symptoms? Are some people more susceptible to the disease than others? "The only people who can really answer those questions are people in Saudi Arabia, where that information resides," Lipkin says.

Mounts has long tried to set up a so-called case-control study in the affected countries to understand what patients have in common. "We need to agree on a series of questions about animal exposure, environmental exposure, and compare the answers with controls of a similar age and the same sex," he says. But the idea has been met with resistance in the region; an early March meeting with scientists and government officials in Riyadh didn't help. "Public health is rarely about the science. It's usually the politics that stand in the way," Mounts says.

Memish, the Saudi deputy health minister, disputes that his country is dragging its feet. He says he's interested in working with any scientist, provided they don't have financial or business interests in MERS. One problem is that the disease has a stigma attached to it, Memish writes in an e-mail: "Nobody wants to take part in anything related to MERS," making it "difficult to get a 6-page, very detailed questionnaire filled by a grieving family member." Some patients' families even moved away to avoid media attention and health officials' questions, Memish writes; questioning enough index cases could take "some years," he adds.

Still, not a single lung section of a MERS patient has ever been published, Frieman notes. "No one has any idea what this thing does in humans," he says. "It blows my mind."

The lack of progress may matter only to a handful of patients and scientists, if MERS continues to remain largely confined to its camel hosts. But SARS, too, lurked in the shadows for years, until it picked up the ability to transmit more easily from one human to the next—and threatened the world with a pandemic. If that happens with MERS, Osterholm says, "we'll all look at ourselves and say 'Why didn't we do more?' "

5. # Building the Ultimate Yeast Genome

1. Elizabeth Pennisi

Chromosome by chromosome, a global army of researchers and students is putting together the first synthetic eukaryote genome.

When geneticist Ronald Davis first suggested a decade ago that his colleagues try to create artificial yeast chromosomes and install them in a living cell, Jef Boeke didn't think much of the idea. Davis, who is at the Stanford University School of Medicine in California, was known as a visionary. He proposed that a lab-made yeast would be the next step in the then-emerging field of synthetic biology. But Boeke couldn't see the point of replicating what nature had already made, especially because designing and synthesizing a 12.5-million-base genome seemed onerous, or even impossible. As Boeke listened to Davis's talk at a major yeast genetics meeting in 2004 in Seattle, he says, "I remember thinking 'Why on Earth would you want to do that?' "

How times have changed. Boeke, a geneticist who recently moved to New York University Langone Medical Center in New York City, and his colleagues have just finished the first complete synthetic yeast chromosome and are well on their way to putting together several more, thanks to technological advances in manufacturing DNA and a global army of collaborators, mainly undergraduate students.

Other researchers have synthesized a bacterium's full genome, but the yeast job is orders of magnitude more complex. For starters, Saccharomyces cerevisiae has 16 chromosomes compared with the one in bacteria. Yet if the effort by Boeke and his army succeeds, it should offer broad benefits. "It gives us the ability to fully explore the yeast genome," Davis says. "If you really want to understand an organism, you should be able to design or redesign one."

Commonly called baker's, brewer's, or budding yeast, S. cerevisiae is vital for many food and drink industries, from beer to bread. Genetic engineers have already tweaked it in myriad ways for many uses, such as making ethanol. Recently it's been put to work building an antimalarial drug, artemisinin, and its potential for churning out other key chemicals is slowly being realized.

Yeast is also a workhorse for biologists probing basic cellular and metabolic processes in eukaryotes. Back in 1996, it became the first eukaryote to have its genome deciphered, and since then yeast geneticists have knocked out every gene and done analyses of all the interactions among them and their encoded proteins. For a long time, it was the only organism in which biologists could readily mutate specific DNA bases, as they found it easily incorporates foreign DNA through a process called homologous recombination.

The synthetic genome under construction by Boeke's army will be the ultimate modification. When it's done, Sc2.0, as some call it, will not be just any ordinary yeast strain. In designing Sc2.0, Boeke and his colleagues streamlined the typical yeast genome and built in sites that will make it possible to reshuffle the genome at will, potentially yielding more desirable, properties and helping biologists figure out what each gene does. The endeavor "is bold, imaginative, and is going to teach us a lot about what the constraints are for synthesizing a whole genome and what constraints there might be relative to genes and chromosomes," says Jasper Rine, a yeast geneticist at the University of California, Berkeley. "It will definitely serve as a landmark in the development of synthetic biology," adds Chantal Shen, a collaborator at BGI in Shenzhen, China.

Until recently, however, no one knew if a truly synthetic chromosome could even sustain eukaryotic life. And when Boeke first attempted to construct a small chunk of yeast chromosome 9—a mere 90,000 bases—few had tried to work with a piece of DNA that big. During the project's earliest days, the effort threatened to fizzle out.

But companies are now able to make ever bigger pieces of DNA, and labs from several different countries are now sharing the labor of synthesizing yeast chromosomes. Those developments, on top of his most recent success, make Boeke hopeful that live yeast containing the whole synthetic genome will be replicating in his lab within 4 years. "We are making history," says Sc2.0 collaborator Junbiao Dai, a molecular biologist from Tsinghua University in Beijing.

## Rough beginning

Boeke traces his change of heart about the synthetic yeast project to 2006, while he was still at Johns Hopkins University in Baltimore, Maryland. Over coffee, his Johns Hopkins colleague Srinivasan Chandrasegaran was trying to convince him to make a large number of zinc-finger nucleases. These DNA-modifying enzymes are Chandrasegaran's specialty, and a toolkit full of them would make the yeast genome easier to manipulate. Boeke wasn't that interested and, almost as a joke, suggested a more drastic way to take control of the yeast genome: synthesize the whole thing. To his surprise, Chandrasegaran jumped on the idea, and the pair brought Joel Bader, a computer scientist at Johns Hopkins, into the discussion.

Despite Boeke's dismissal of Davis's proposal just 2 years earlier, the trio quickly concluded that making an artificial yeast genome would, in fact, be worthwhile if it could be a testbed for learning about the genome itself. They decided to start with the 90,000-base "R" arm of chromosome 9—the shortest in the yeast's genome—and spent a year arguing about how the synthetic sequence should differ from the natural one. The trio considered just including the genes they wanted, "but we quickly realized it would be very risky to eliminate whole bunches of genes" without really knowing in advance what the effect of that loss on the whole system would be, Boeke recalls.

So they started with the natural sequence of the chromosome 9 arm and instead added DNA to it that would enable them to induce changes at will. They inserted short sequences of DNA called loxP just after the end of each nonessential gene—those they knew could be knocked out or changed without killing the yeast. They also put loxP sites near significant landmarks, such as the telomeres at the tips of chromosomes and the centromeres at each one's center. LoxP is a part of a standard molecular biology tool. When activated by a chemical added to cells, it kicks off a chromosomal version of musical chairs. The result: genomic rearrangements and new yeast strains with different properties. Boeke and his colleagues called this system SCRaMbLE, for synthetic chromosome rearrangement and modification by loxPmediated evolution.

Although Boeke's team wanted to make the genome unstable when they desired, they didn't want the genome to undergo changes or rearrangements of its own accord, potentially disrupting the integrity of the synthetic strain. To increase the genome's stability, they took out mobile DNA elements, such as retrotransposons, that in theory could jump to new spots at any time.

The design they pioneered with the chromosome 9 arm cuts out other noncoding DNA as well. The natural telomeres, the repetitive regions next to the ends of chromosomes that can also be unstable, are now gone. Shorter synthetic ones will cap each chromosome. The researchers also took out many of the introns, the noncoding sequences between coding regions of a gene.

The quest for stability also prompted some radical steps. The team is taking out the DNA coding for the yeast's 275 transfer RNAs, which shuttle amino acids to the ribosome for stitching together into a protein. The transfer RNA genes are essential, but because their sequences can sometimes cause a cell's DNA copying machinery to stall out, they are "DNA damage hotspots," says Patrick Yizhi Cai of the University of Edinburgh in the United Kingdom. Cai is building a "neochromosome" that will have all those genes relocated onto it. Collected into one place, these genes will still be available to the modified organism, but will do less damage if they become unstable. (The neochromosome will not increase the total number of chromosomes in Sc2.0, because Boeke plans to do away with chromosome 1. It naturally has just 230,000 bases, and given the design rules of Sc2.0, its synthetic counterpart would be as much as 70,000 bases shorter. Worried that this diminished chromosome would be unstable, the team plans to append that DNA to another synthetic chromosome.)

Boeke and his colleagues added two more modifications to the design. Throughout the genome, they are inserting short, specific DNA sequences, detectable by the polymerase chain reaction, that distinguish each synthetic chromosome segment from its natural counterpart. Finally, they tinkered with some of the natural stop signs in eukaryotic genomes—the "stop" codons that tell a cell when to cease making an RNA. On the chromosome 9 arm, the researchers turned every single instance of one stop codon, "TAG," into another, "TAA," by switching in the base adenine (A) for a guanine (G). In the complete synthetic genome, they will make more than 1000 such substitutions all together. The "stop" sign is therefore still there, but in a different form. But this frees up "TAG" as a codon for an artificial amino acid, if they ever decide to introduce one into the makeup of their enhanced yeast.

With the aid of a software program developed by computer scientist Bader, Boeke designed a version of the chromosome 9 arm incorporating all of these changes and carefully checked as much as possible that the added bases would not interfere with the expression of any remaining yeast genes. He then contracted with a biotech company called Codon Devices to synthesize the chromosomal arm.

Eleven months went by with no word from the company, which had never attempted to make such a long piece of DNA. "That was a tense time," Boeke recalls.

But that dark period proved inspirational as well. Boeke wondered whether he could speed up and decrease the cost of the work, as well as help others learn molecular biology, if he set up a course dedicated to building a synthetic yeast genome (see sidebar, p. 1429). He launched the idea as a summer school offering in 2007, even before he knew that the 9R synthetic arm he had ordered would work. Now, 6 years later, the thrice-weekly course at Hopkins is packed, even on Friday nights. "We were overwhelmed with interest" from biology, engineering, computer science, public health, and other majors, says Boeke, who, despite his move to New York, will keep the Hopkins course going with colleagues there.

## Scrambled DNA

Once Codon Devices finally delivered a 90,000-base circular chromosome, it took Boeke and colleagues months more to successfully stick it into the yeast, cut out the natural 9R, and then test the effects. The synthetic chromosome arm performed without a hitch, yielding healthy yeast and reasonable gene expression, Boeke's team reported in 2011 in Nature.

The SCRaMbLE system worked as well. The yeast carry a piece of DNA that codes for a highly modified version of an enzyme called Cre recombinase, which randomly deletes or inverts the DNA lying between any pair of loxP sites. This enzyme is usually stuck in an unfolded form in the cell's cytoplasm, but when the researchers add the chemical estradiol to yeast, it folds up and can sneak into the nucleus. LoxP activation leads to the random removal of different genes, sometimes killing the yeast but other times simply changing the yeast's properties, rendering it incapable of making certain amino acids, for example. "We knew our design had panned out," Boeke says. "Our hope then was to scale up."

Meanwhile, the students from that initial summer course and subsequent ones had taken on chromosome 3. From an initial class goal of 1500 bases per student, productivity had ramped up the point where each student promised to turn in at least 30,000 bases by the semester's end. It took 49 students and 1.5 years to make the synthetic chromosome's 272,871 bases—the native version has 316,667 bases—but under the guidance of Boeke's postdocs Narayana Annaluru and Héloïse Muller, their effort has paid off with a publication online this week in Science (http://scim.ag/NAnnaluru). Yeast carrying the shortened, modified chromosome grew normally and looked little different from their natural counterparts under almost all the growing conditions tested, the researchers report.

"They made some pretty dramatic changes," says Mike Tyers, a yeast systems biologist at the University of Montreal in Canada. "But on the other hand, they were conservative enough that the experiment would have a chance of working. I think they hit the sweet spot."

As Boeke and his colleagues entered the homestretch with chromosome 3 and began to work on other yeast chromosomes, a chance meeting between Cai, then Boeke's postdoc, and Ying-Jin Yuan of Tianjin University in China at a synthetic biology competition led to the globalization of the effort. "An international project wasn't on our mind at all," Boeke recalls. Yet Yuan was so excited about Sc2.0 that he got Huanming Yang of the sequencing powerhouse BGI involved. Yang helped organize the first synthetic yeast genome meeting, held in Beijing in 2012, and partnerships emerged. Yuan set up his own "Build A Genome" course and in the summer of 2012, 60 Chinese students assembled all the bases needed for chromosome 5.

Momentum built elsewhere as well. Tom Ellis, a synthetic biologist at Imperial College London, became hooked on the ambitious venture after attending the Beijing meeting. He helped organize a second Sc2.0 meeting in July 2013, following a larger synthetic biology conference at which the British government announced it would commit £1 million toward the synthetic yeast genome project. Other countries are getting involved as well, Boeke says (see chart).

The hope is that in 2 years, the various partners will have stitched together each of the yeast's chromosomes, with strains containing one chromosome each. Then Boeke will tackle the difficult task of putting them all in one organism. "It's a little surprising that we can have this grand idea with so many moving parts" and multiple organizations making different chromosomes, says Boeke's senior lab coordinator, Katrina Caravelli. But so far, "it's working well."

## Custom yeast

Once the synthetic organism is in hand, yeast biologists will be eager to tinker with it. With 5000 loxP sites, the synthetic genome will be ripe for mutations, opening the way for researchers to take a systems approach toward understanding which genes matter when during the yeast life cycle.

The longer they expose a yeast strain to estradiol, the greater the number of genetic changes that the SCRaMbLE system will induce. Leslie Mitchell, a postdoc in Boeke's lab, plans to grow the mutated strains under different conditions, document their behavior and other characteristics, and sequence their genomes. From the data, she hopes to piece together how different genes interact.

SCRaMbLE should also make it easier to harness yeast for biotechnology. Right now, finding a yeast strain that makes enough of a product to be commercially viable "takes many steps and many person years," Boeke says. SCRaMbLE should speed that search up by providing an "unbelievable number of mutants that would be much more time-consuming to get one at a time." With more strains to evaluate, researchers are more likely to find the best one for the job.

Success at synthesizing versions of yeast's native chromosomes will also open the way to adding entirely novel chromosomes to the organism—ones that endow it with brand-new properties or enable it to model human diseases. Boeke, for example, would like to install in yeast all of the genes of the molecular pathway that, in humans, is defective in Lesch-Nyhan syndrome, a rare disorder characterized by gout and kidney and neurological problems, including self-mutilating behavior. By modeling the molecular defect in yeast, he hopes to figure out how the mutation affects eukaryotic cells in general.

Researchers who have watched the steady progress made by Boeke's army say the project will be a boon for basic science, promising "deeper mechanistic understanding of biological processes in yeast," says Huimin Zhao, a synthetic biologist at the University of Illinois, Urbana-Champaign.

But Kirsten Benjamin, a synthetic biologist at Amyris Inc., the Emeryville, California, company that harnesses yeast for producing artemisinin and other chemicals, expects that problems loom for the project: The more synthetic DNA Boeke tries to incorporate in a yeast, the sicker the strain will likely be, she predicts. "We're going to find a bunch of ways where it doesn't work," she says. But she agrees that the problems could be revealing, saying they "will allow us as a scientific community to discover all these unappreciated phenomena."

She and others are not sure how useful synthetic genomes will be for commercial applications. Less drastic approaches to improving yeast's manufacturing prowess may work better. "From a practical viewpoint, it is too costly to [make synthetic genomes] for most engineering applications," Zhao says.

But maybe it's enough to just build a eukaryotic genome once, Boeke suggests. "In a way, you can put it as a kind of milestone, like the first human genome was a milestone for genomics," he says. "When we finish it, we can really plant a flag in it."

6. # Student Assembly Drives Yeast Project

1. Elizabeth Pennisi

For two undergraduates, helping build a synthetic yeast genome proved much more than just another lab course.

The homework that James Chuang and Katrina Caravelli turned in to Jef Boeke consisted of just four letters: A, C, G, and T, representing DNA's four bases, each repeated thousands of times. But there was nothing tedious about their assignment: “Build A Genome,” as the undergraduate course's name put it. Boeke conceived the course at Johns Hopkins University in Baltimore, Maryland, 6 years ago while struggling to figure out how to do all the work required to build a synthetic yeast genome (see main story, p. 1426).

For Boeke, the course supplies labor—scores of undergraduates have taken it so far. For students, it offers intensive training in synthetic biology and the thrill of taking part in frontline research. “I was really fascinated by the potential and by my ability to have an impact,” recalls Caravelli, who signed up for the course in 2008 and is now Boeke's senior lab coordinator at New York University. During the semester-long course, students learned basic molecular biology procedures such as performing polymerase chain reactions and cloning DNA in bacteria.

Each student committed to completing 10,000 DNA bases during the course. “My building blocks went to chromosome 8,” Chuang, now a graduate student in biomedical engineering at Boston University, says proudly. After being supplied with the DNA sequence he needed to synthesize, he started out with 16 pieces of DNA about 75 bases long, ordered from a commercial DNA synthesis firm. The ends of some pieces overlapped, so when he mixed the pieces together with enzymes, they self-assembled into 750-base spans dubbed building blocks. After making sure the building blocks had the correct sequence, he put them into bacteria to generate many copies of the newly assembled DNA. Now, Johns Hopkins graduate student Jingchuan Luo is putting those bigger DNA sections into yeast and making “minichunks” about 3000 bases long. If the yeast stays healthy, then she adds the next chunk in line to it, and so on. Her goal: to make a yeast strain with a totally new chromosome 8.

Caravelli recalls that her own bacteria sometimes wouldn't grow with the yeast segment in their midst. She enjoyed figuring out why. “The trial-and-error process challenges you to think properly like a scientist.”

Boeke's course has proved such a success that three other U.S. universities are hosting or will soon host their own. Because it's now cheaper to buy 750-base segments than to have students make them, current students start with such DNA blocks and turn them into 3000-base spans, and, ultimately, 10,000-base chunks. Class productivity has soared. Each student's target is now 30,000 to 50,000 bases.

The students have high hopes for their work. “I want to see synthetic biology do really useful things for society,” Chuang says. “The synthetic yeast genome provides a template for doing [that].”

7. Breast Cancer

# Breast Cancer: A World of Differences

1. Kelly Servick

Advances in detecting and treating breast cancer offer an ever brightening outlook for women diagnosed in wealthy countries, but the recent progress has been far from uniform, and in poor countries mortality remains disproportionately high.

Click the image for a larger version.

[CREDIT: SOURCES (top to bottom): IARC'S GLOBOCAN 2012; NATIONAL CANCER INSTITUTE]

8. Breast Cancer

# Dare to Do Less

1. Eliot Marshall

Scientists are looking for ways to spare women from aggressive treatment of ductal carcinoma in situ, a diagnosis that only sometimes leads to invasive breast cancer.

Shelley Hwang, a surgeon who has treated women with breast cancer for 17 years, is troubled by the thought that many who have gone under her scalpel really didn't have cancer. What they had, she says, was irregular tissue that may increase the risk for cancer. Not knowing much about these abnormalities, however, oncologists decided decades ago that the right thing to do was to remove them. That's still being done.

A tidal wave of such ambiguous cases began to pour into clinics in the early 1980s. They were the product of a drive to catch cancer early, aided by new breast imaging methods that found lumps or tissue aberrations that would not have been noticed before. Hundreds of thousands of patients were told that they had "ductal carcinoma in situ," or DCIS—cancer confined to a milk duct. It has also been called "stage zero" cancer. In the past, these women typically received mastectomies, followed by radiation and drugs.

Hwang, now at the Duke University School of Medicine in Durham, North Carolina, says that, with no decisive evidence, oncologists felt they had to treat each DCIS case as if it were invasive cancer. "We were removing all these breasts" to take out DCIS lesions, "the majority of which might never become clinically significant," she says. Today, the diagnosis of DCIS usually leads to less radical surgery—removal of a few cubic centimeters of tissue (a "lumpectomy"), followed by radiation and hormone therapy. The cohort of U.S. women living with the diagnosis has risen steadily; one forecast estimates they will number 1 million by 2020.

But Hwang and other oncologists worry that women are still overtreated for DCIS that would never become life-threatening. They hope it will become possible to do more sophisticated analysis of each DCIS patient's risk for invasive cancer and adjust treatment accordingly, avoiding radiation treatment, for example, or even in some cases surgery. Some, like oncologist Laura Esserman of the University of California, San Francisco (UCSF), have argued for years that the diagnosis should have a gentler name, one that omits "carcinoma." She proposes calling these and similar slow-growing tissue irregularities "indolent lesions of epithelial origin," or IDLE. The goal, Esserman says, is to let doctors and patients "take a step back" and be less aggressive with therapy.

Five years ago, leading physicians and researchers met at the Bethesda, Maryland, campus of the National Institutes of Health to review what's known about DCIS. They concluded in a consensus document that it would be worth considering a less "anxiety-producing" name, as well as "less therapeutic intervention" if it could be done without increasing the risk of subsequent cancer. Testing new approaches to treatment is difficult, given that current practice is judged a success: Ninety-eight percent of U.S. women treated for DCIS die of something else. Yet even this small risk can be lowered with postsurgery radiation, which reduces the chances of subsequent invasive cancer by 50%, according to a 2011 study led by Irene Wapnir of Stanford University in California. Few people may try the experimental therapy when the norm looks so good. But Esserman and Hwang are running trials in which women diagnosed with DCIS opt out of some parts of standard therapy.

To guide such treatment, Hwang says, "we need to have better predictors of which DCIS will likely become invasive and which won't." Progress has been slow, but a U.S. company—Genomic Health Inc. (GHI) of Redwood City, California—launched a test in December 2011 called DCIS Score, which monitors seven cancer genes in DCIS tumors to rate the risk that they will become invasive. Many physicians argue that its predictive value is small, and several university-based groups claim to have molecular markers that are better for detecting certain high-risk types of DCIS.

Still, both Hwang and Esserman describe the DCIS Score test as a useful first step. Such tools will help women decide which DCIS cases to wait and watch over, Esserman says. "We have to put just as much effort into making sure we don't overreact" to the fear of cancer, she says, as we put into treating it.

## Fear is the driver

"We didn't have much DCIS in the United States until we got into mammography screening," says Joann Elmore, an oncologist at the University of Washington, Seattle. Now, Elmore says, "we are seeing little calcifications, little white dots [on breast scan images]. We don't want to miss anything," especially because failure to detect cancer is "the number one cause" of malpractice allegations. When doctors do spot an anomaly, they are likely to ask for a biopsy.

For more than 60,000 women per year in the United States, that leads to a diagnosis of DCIS. The diagnosis is not new, but Hwang and others believe that the lesions now diagnosed as DCIS may differ from those with the same label in the 1940s and 1950s. They are smaller and, unlike earlier ones, most are not palpable. Under a microscope, Hwang says, the cells in the DCIS lesion look very similar to cells in an invasive breast cancer and are scored on three grades of abnormal appearance like those used for invasive cancer. But they are contained within the milk duct and may remain there safely for a lifetime. It's not known what enables some to escape. But it is clear that some do—and become dangerous.

The reported U.S. incidence of DCIS has increased dramatically over the past 3 decades, especially among women over 50. Estimates of the fraction of women diagnosed with DCIS who might go on to develop invasive cancer without treatment range from 14% to 50%. But because almost all cases are treated to prevent invasive cancer, it is difficult to get firm data. Pathological and molecular analyses of biopsies have already shown that some types of DCIS are more likely to be invasive, others far less, Esserman says. Yet even as doctors in the United States found and treated far more DCIS, the incidence of invasive breast cancer has remained fairly steady. To some, this suggests that treating all DCIS does not do much to stop more dangerous cancers. This was the conclusion of a 2011 computer modeling study of cancer trends from 1978 to 2003 by Elissa Ozanne, then at Massachusetts General Hospital in Boston; Esserman; Hwang; and three others.

## Forgoing the knife

A few clinicians have begun to offer experimental ways of managing the disease. At Duke, for example, Hwang leads a cooperative trial at 23 U.S. sites that offers some women with DCIS an opportunity to try hormone therapy alone. Only postmenopausal women are enrolled, and only if their DCIS is rated low-risk based on traditional pathology measures and their lesions are estrogen-dependent. They are monitored closely to see if the DCIS starts to recede. Hwang hopes the trial, which ends in 2015, will establish that it's possible to treat low-risk DCIS without surgery.

Esserman and colleagues at UC are setting up clinical trials to offer some women with DCIS a chance to enroll in a regime of "watchful waiting" and reduced treatment, similar to that offered to men with prostate cancer judged to be low-risk. They rolled out a plan they call the Athena Breast Health Network in 2009, linking five UC medical centers. Esserman says the network is developing new classifications of breast lesions, using DCIS Score in combination with standard pathology to rate the chances that patients might develop invasive cancer. Patients judged to be high-risk will be treated as in the past, but others will be offered a choice of going without radiation, or even skipping surgery. They will be monitored closely. There are "plenty of people" who are willing to live with some uncertainty, Esserman says, and "we should not browbeat them into having treatments" that may not benefit them.

## Testing times

As cancer physicians and their patients wait for results, women are left with little to guide them if they wish to avoid surgery, radiation, and drug treatments. Beyond the standard biopsy and personalized risk analysis based on genetics, they do have GHI's test, certified by the U.S. Centers for Medicare & Medicaid Services.* Priced at $4380, DCIS Score is a modified version of Oncotype DX, the test the company has sold for a decade as a way to classify the aggressiveness of invasive tumors. DCIS Score probes tissue samples taken during breast biopsies for the activity of seven genes linked to cancer and five other reference genes. (Those genes include Ki-67, STK15, Survivin, CCNB1 [cyclin B1], MYBL2, PR, and GSTM1.) Based on gene expression levels, an algorithm calculates whether a woman who has already had surgery (but not radiation) for DCIS is likely to see the lesion come back or even develop into invasive cancer. Lawrence Solin, an oncologist at the Albert Einstein Medical Center in Philadelphia, Pennsylvania, led the first and, so far, only published effort to validate DCIS Score. With support from GHI, the U.S. government, and the Breast Cancer Research Foundation, Solin's group retrospectively analyzed tissue from 327 DCIS patients who had undergone surgery but not radiation. The team reported in 2013 that the three levels of risk scores the test gave for tissue samples correlated well with patient outcomes. Women judged to have a low DCIS Score, for example, turned out to have only a 3.7% risk of developing invasive cancer within 10 years. Those with middling scores had a risk of 12.3%. And the highest scoring group had a risk of 19.2%. According to the authors, in their 327-patient sample, the gene test forecast risk better than current pathological methods, which rely mainly on the analysis of cell structure and physiology in biopsied tissue. Many researchers aren't wowed by the test, however. Karla Kerlikowske, an oncologist and epidemiologist at UCSF, calls DCIS Score "OK," saying that at least it doesn't exaggerate risk. But she thinks that it doesn't flag some of the most dangerous subtypes of DCIS, which are known to exist but are not fully defined. She and a group of researchers at UCSF have been analyzing additional potential markers of high-risk DCIS, which they would like to combine in a test. For example, they would include the activity of genes p16 and COX-2, which are not in GHI's test. Kerlikowske says the UCSF team has run comparative checks and found that DCIS Score "misses about half the invasive cancer" their method finds. The results are unpublished so far, however; Kerlikowske says she failed to win funding for a proposed full head-to-head comparison of the two assays. A company that was interested in the test 2 years ago has not yet moved ahead with it, she says. Pathologist Agnieszka Witkiewicz and researcher Erik Knudsen of the University of Texas Southwestern Medical Center in Dallas have flagged a gene set that overlaps with UCSF's but also includes the well-known oncogenes RB and PTEN. "RB is probably our favorite," Knudsen says, because it gives more prognostic information in the group they've examined—DCIS patients from a Philadelphia clinic who had surgery but no radiation. He claims that this index "outperforms" DCIS Score in spotting the risk for certain types of invasive cancer. "If money were no object and I had a staff of thousands," Knudsen says, he would try to develop it. Another DCIS risk assay still in the earliest stages of study focuses on protein levels in the duct's structure versus protein levels in the tumor. DCIS becomes invasive only when the duct gives way. In laboratory studies, Michael Allen and Louise Jones of the Barts Cancer Institute at Queen Mary University of London found that a protein known as integrin αvβ6, involved in cell signaling and attachment, was present at higher levels in the duct's myoepithelial cells when DCIS had become invasive. They report that this causes these ductal cells to change from tumor suppressors to tumor promoters. And they found a similar high level of αvβ6 in breast tissue from DCIS patients with invasive cancer. But Allen acknowledges that before these insights can be translated into a practical test, they must be tested in a clinical trial—and that would be difficult to carry off for many reasons. Until these would-be rivals standardize their methods and carry out trials, says Steven Shak, executive vice president of R&D and co-founder of GHI, their claims must be taken as interesting but not proven. Shak, who is a co-author on the Solin paper, claims that DCIS Score is "widely used." He adds that it is being validated in a second, independent patient group, although he declines to "upstage" the researcher who's running the trial by releasing details. Competition for all the test designers may be on the horizon. The U.S. Department of Defense's Breast Cancer Research Program has proposed to aid the hunt for better DCIS risk markers. So has the U.S. National Cancer Institute, which plans to spend about$5 million a year on such work, according to the director of its cancer prevention division, Barnett Kramer.

The help can't come fast enough, says Fran Visco, an attorney who heads the National Breast Cancer Coalition, an advocacy group in Washington, D.C. "We keep running down these roads putting a lot of time and money behind things but … we don't have the basic information that we need." She gets calls from women "all the time" asking what they should do about a DCIS diagnosis. Visco tells them to study the data and balance the risks and benefits for themselves, because, "we don't really know the answer."

* Correction (22 April 2014): This article incorrectly reported that a genetic test for ductal carcinoma in situ sold by Genomic Health of Redwood City, California, was approved by the U.S. Food and Drug Administration; the test, known as DCIS Score, is certified by the U.S. Centers for Medicare & Medicaid Services.

9. Breast Cancer

# The 'Other' Breast Cancer Genes

1. Sam Kean

Since the discovery of BRCA1 and BRCA2, dozens more breast cancer genes have come to light. But what risk they pose—and what to tell women who carry them—remain quandaries.

A quarter-century after the first breakthroughs in breast cancer genetics, scientists have a good grasp on the risks that come with mutations in BRCA1 and BRCA2, the most prominent breast cancer genes. Frustratingly, though, just as the sky began clearing with the BRCAs, a whole flock of other genes has swept in and muddled the view.

BRCA1 and BRCA2 dominated the breast cancer agenda for so long for good reason. BRCA cancers attack women early in life, they're aggressive and deadly, and they might hit three or four women in one family, a devastating legacy. On top of all that, they've been embroiled in political controversy, as the subject of a landmark Supreme Court case about the legality of patenting genes.

However infamous, though, the BRCAs account for only about 25% of breast cancers with a hereditary component. And with the rapid expansion of next-generation DNA sequencing, scientists have now unmasked dozens of other genes associated with breast cancer. PALB2, ATM, CHEK2, and others probably won't gain the notoriety of either BRCA gene, but they've already become important players. "We always knew we should [test for] 10 genes instead of two," says Charles Perou, a geneticist at the University of North Carolina (UNC), Chapel Hill, "but it wasn't technically feasible in the past. Now it's practical."

In theory, women at high risk of breast cancer might benefit from such testing. They and any female relatives carrying a risky mutation could undergo screening more often, increasing the chances of catching tumors early. If the mutation looks especially risky, they might opt for prophylactic mastectomies, surgery to remove their ovaries, or sometimes both, because breast cancer and ovarian cancer share genetic risk factors. And if they do develop tumors, knowing the underlying genetic basis may help determine treatment.

But as Matthew Ellis, a geneticist at Washington University in St. Louis, puts it: "As the genetic diagnosis gets easier, the interpretation gets more difficult." Identifying a risky mutation in these other breast cancer genes has proved challenging. Geneticists simply don't know in many cases which mutations within a given gene will lead to cancer and which won't. A clearer picture will emerge only from long, involved trials to link mutations to clinical diagnoses.

This has led to a lot of hand-wringing among doctors and genetic counselors about what to do in the short term: when and whether to test the full panoply of other breast cancer genes in their patients, and how much to reveal to women who are tested. Some in the field even suggest not telling patients about certain mutations they harbor in their own DNA until geneticists can assess the risk more accurately, however long that takes.

Depending on what the tests turn up, most geneticists recommend that patients seek genetic counseling to help them understand the risks revealed by the tests. But not all women receive such counseling, especially in the United States, where testing companies can market directly to family physicians.

And even the best counseling can't alleviate the fundamental problem that the data continue to outpace the understanding. This has left doctors and patients alike in a bind, Ellis says. "You have to say ['I don't know'] a lot," he says, "and that's a pretty unsatisfactory answer."

## Repair crew

Biologically, the role of the other breast cancer genes is becoming clearer. Like the BRCAs, most of them help repair broken DNA, says Mary-Claire King, who mapped BRCA1 in 1990 and is a geneticist now at the University of Washington, Seattle.

Specifically, these genes help repair double-strand breaks. If a single strand of DNA's double helix breaks, cells can repair the flaw pretty easily by using the other, complementary strand as a template. Double-strand breaks are messier, and cells often have a tough time aligning the tattered fragments properly. Repair work therefore requires specialized machinery, and dozens of genes lend a hand.

If a woman inherits a mutation in one of these genes, her cells can get along fine at first, because every cell has two copies of each gene, one on each chromosome. Problems arise only if the good copy gets disabled as well. Unfortunately, many genes involved in breast cancer have a high likelihood of getting shut down, King says, because they reside in "bad neighborhoods" on their chromosomes. For example, they might sit near a high concentration of Alus, mobile genetic elements that have a nasty habit of inserting themselves into genes and disrupting function.

If both copies of a DNA-repair gene end up disabled, the cell can lose the ability to repair double-strand breaks. At that point, King says, "all kinds of hell breaks loose" and breast cancer will likely result.

But geneticists still struggle to say which mutations in these new genes are important risk factors in cancer. Marc Tischkowitz, a geneticist at the University of Cambridge in the United Kingdom, sums up the difficulty succinctly: There is no BRCA3, he says. That is, no other single gene, when mutated, can explain a large number of breast cancer cases.

Consider PALB2, which interacts with BRCA2 in repairing double-strand breaks. PALB2 mutations cause a two- to threefold increased risk of cancer, a significant jump. (For comparison, harmful BRCA1 mutations increase the risk of cancer by about fivefold; harmful BRCA2 mutations increase it by about fourfold.) But PALB2 mutations appear so rarely that they probably don't explain more than a tiny percentage of tumors. They're so rare, in fact, that Tischkowitz had to organize an international consortium to study the gene, because no one country had enough cases. So while PALB2 and other risky but rarely mutated genes contribute to our understanding of breast cancer, they explain few cases of breast cancer overall.

Adding to the difficulties, some mutations in the newly identified genes have incomplete penetrance. That means they only sometimes lead to cancer, making them tricky to interpret. If a woman has two or three low-penetrance mutations spread among a few different genes, scientists also don't know in most cases whether the risks add up in a straightforward way.

Some mutations are clearly worse than others, King says. As examples, she mentions mutations that lead to premature stop codons during protein production, as well as insertions or deletions that lead to frameshift mutations. "Critical mutations" like these, she says, "whack the gene completely."

Unfortunately, most mutations are more ambiguous. A point mutation near a functional domain, for instance, or a mutation in a noncoding region might well hamper a gene—but then again might not. Equally daunting, geneticists see huge numbers of different mutations in the population at large, each of which requires independent evaluation. In a phrase that pops up over and over—it's practically a mantra in breast cancer research—geneticists refer to these ambiguous mutations as "variants of uncertain significance."

Scientists can now explain the genetic roots of about half of all family clusters of breast cancer, Tischkowitz says. "About a quarter of them will have mutations in BRCA1 and -2, and a quarter of them will have a combination of common, lower penetrance variants. But about half of them are still unexplained. Which is a puzzle for us."

And things may get worse before they get better. Perou says that preliminary research has identified on the order of 100 other genes that might—or might not—also contribute to breast cancer. Sorting out the rogues from the pretenders, though, "is going to take some time," he acknowledges. "It's much easier to make a discovery than to show that that discovery has clinical utility."

## In the clinic

Current tests for breast cancer reflect this uncertainty. "Six years ago, when we had our BRCA1 and -2 results in hand, for most intents and purposes, we were done," says James Evans, a professor of genetics and a genetic counselor at UNC Chapel Hill. But particularly in the past year, so-called expanded panels have come onto the market. These genetic tests scour for mutations in a dozen or more breast cancer genes. As a result, Evans says, especially with women whose families have a history of breast cancer, "we are now faced, daily, with decisions about whether to expand our reach in testing."

Adding to the challenge, the panels differ from company to company, sometimes greatly, and the information they provide is not always helpful, he notes. He stresses that panels do have their use. "But the field has not settled on consistent, evidence-based guidelines," he says, "that tell us when we should use an expanded panel, when we shouldn't, and even what genes should be on that expanded panel."

To compound the problem, some genetic variants that predispose, say, Caucasian women to breast cancer might not predispose black or Hispanic women, and vice versa. So in addition to the general clinical trials necessary to link variants to an increased risk of cancer, scientists might also need to conduct separate clinical trials on different ethnic groups to sort out which mutations pose the worst risks for each.

Given all the uncertainty, geneticists and genetic counselors disagree about how often to use expanded panels, and especially about how much information they should pass along to patients. They do generally tell patients about unambiguously harmful mutations, and advise them to step up surveillance for early-stage tumors. But many geneticists hesitate to pass information about ambiguous mutations to patients. "I think it's probably better for them not to hear that," Perou says. "It's confusing to me, it's confusing to them, and to be accurate, you would have to report a million variants for every person. That's completely not helpful."

Faulty testing can also affect family members. In the 1990s, Tischkowitz says, preliminary studies strongly linked CHEK2 to breast cancer; later studies reduced the risk substantially. But imagine that a geneticist had told a woman that a CHEK2 mutation probably explained her family's history of cancer. Some of her sisters or daughters could easily test positive for the mutation as well.

In cases like this, Evans says, "you've just assigned a whole bunch of people in the family to screening and procedures that they didn't need." Perhaps worse, other family members, who tested negative for the mutation, might believe themselves off the hook. "You've given them false reassurance," Evans says, "and they haven't gotten the extra surveillance that they would benefit from."

Above all, geneticists and counselors worry about inadvertently pushing patients to take drastic steps. "Patients will go to the end of the earth to protect themselves from breast cancer," Ellis says, including prophylactic surgery. Mastectomies are still vastly more common among women with BRCA mutations, but a small percentage do elect for the surgery because of non-BRCA mutations.

So far, these cases seem limited to mutations with PALB2 and other genes with rare but clearly harmful variants. Still, geneticists worry that, as more and more genes are linked to breast cancer, women might opt for mastectomies based on faulty or incomplete information, like the early CHEK2 studies.

"One can imagine the headlines of people having unnecessary preventative surgery, when later on it's discovered that the risk of these genes may not be as strong," Tischkowitz says. He adds, "Maybe we'll look back in time and think we were overcautious, but I'd rather it be that way than the other way around."

10. Breast Cancer

1. Jocelyn Kaiser

For more than 2 decades, Fran Visco, president of the National Breast Cancer Coalition, has been a force behind the second biggest U.S. breast cancer research program.

Frances Visco may be the most influential nonscientist ever in the field of breast cancer research. Her grassroots patient advocacy group, the National Breast Cancer Coalition (NBCC), has a small staff and does not run a research foundation. It has no scientific advisory board, although "we talk to scientists all the time," says spokeswoman Michelle Zelsman. Yet the coalition has had an out-sized influence on how breast cancer research money is spent. Twenty-two years ago, the group convinced Congress to put $210 million into a new breast cancer research program in an unusual place—the Department of Defense (DOD). Since then, that program has awarded nearly$3 billion in grants, making it the second largest funder of breast cancer research in the United States after the National Cancer Institute (NCI).

Visco has sat on the oversight board from the beginning. Her group has pushed for high-risk, high-reward research and studies that affect cancer patients' lives. Many say the DOD program has been a big success, drawing new talent into the field and launching research that might not have made it through NCI review.

But Visco is not satisfied, noting that, in her group's view, the decline in mortality from breast cancer over the past 2 decades is not significant. That's why 4 years ago her coalition made another unusual move: It vowed to "end breast cancer by 2020" by attracting scientists to pursue what some say are wildly ambitious goals, such as a preventive cancer vaccine. For a group that prides itself on embracing evidence-based medicine, it was a surprising turn.

In a recent interview at the coalition's office in downtown Washington, D.C., Visco discussed how its thinking has evolved since 1992. "In those days, we talked in terms of 'We just need to find who the best scientists are, the most creative and make sure they have the money they need to do what they do,' " Visco said. "Maybe that was very naive."

## Call in the Army

Visco left a law practice to help launch NBCC after being diagnosed with breast cancer at age 39, joining other breast cancer activists including epidemiologist Kay Dickersin of Johns Hopkins University in Baltimore, Maryland, who had also had breast cancer, and breast surgeon Susan Love. They followed the lead of AIDS activists, who had shown that patients could be a powerful force in boosting funding for their disease. In fall 1991, the newly formed coalition issued a call to supporters that drew 600,000 letters asking Congress to increase funding for breast cancer research. The group then held its own "research hearings" on Capitol Hill with handpicked scientists and concluded that to fund all promising ideas, NCI needed another $300 million a year. Senators Tom Harkin (D–IA), chair of the Senate appropriations subcommittee that funds the National Institutes of Health (NIH), and Alfonse D'Amato (R–NY) agreed to boost NCI's then–$133 million budget for breast cancer research by $64 million. They also put another$210 million in the DOD budget, which already had a small women's health research program, with the understanding that NCI would control how the money was spent.

But Visco's group changed its mind after meeting with then–NCI Director Samuel Broder, who said "we can't turn a huge battleship on a dime," Visco recalls. She says her group had a "whole different experience" at the U.S. Army, where a general agreed the money should go to innovative research and that advocates should have a seat at the table. "We decided we wanted to keep it at the DOD," Visco says.

To the relief of wary scientists, the Army asked the Institute of Medicine (IOM) for advice on how to spend the money—then expected to last just 2 years. The committee, which included Dickersin and virologist Harold Varmus, a Nobel Prize winner who soon became director of NIH, suggested that most of the money be spent on peer-reviewed basic research.

In a departure, IOM also recommended that the program's steering committee, known as the Integration Panel, include advocates; the Army later added advocates as voting members of peer-review panels despite qualms from some scientists. The Integration Panel, the Army decided, would also have unusual discretion: Unlike institute councils at NIH, it doesn't award grants based largely on peer-review scores but takes a "more hands-on" approach, Dickersin says. It can also take programmatic interests into account—for instance, it can reject a proposal with a stellar scientific score if it feels the program has already funded enough grants in that area.

Thanks to the coalition's lobbying, Congress has funded the DOD Breast Cancer Research Program every year since 1993, although not at the rate of growth of NCI, which now spends about $600 million a year on breast cancer. The DOD program now receives between$120 million and $150 million a year. Nor are the grants easy to get: Success rates average 13% over 20 years, compared with 23% at NCI. "A lot of people just look at it as another source of money," Dickersin says. But she and others argue that advocates trained in the peer-review process have pushed the program in a different direction than NCI. They add "a sense of urgency and passion as well as an important perspective on the feasibility and ultimate impact" of a proposal, says Gayle Vaday, a program manager with the DOD program. And because the DOD program has to spend all its money each year, unlike NCI, it can "move on a dime" and quickly launch new mechanisms or respond to new opportunities, says breast cancer researcher Dennis Slamon of the University of California, Los Angeles. In another review in 2004, IOM found "pretty strong consensus that DoD was adding value, not just replicating what NCI would otherwise do," says research professor of public policy Robert Cook-Deegan of Duke University in Durham, North Carolina, a member of that panel. Many of the program's successful projects would have been unlikely to receive funding from NCI, researchers say. Slamon received one early grant to collect tumor tissue samples that firmed up his group's contention that some women's breast tumors carry extra copies of a gene coding for a surface protein, HER2, which makes the cancer particularly aggressive. NCI wasn't interested in funding the tissue bank because it wasn't hypothesis-driven, Slamon says. He adds that the studies helped persuade Genentech to move ahead with clinical trials of Herceptin, an antibody targeting HER2 that is now standard of care for this tumor type. Cold Spring Harbor Laboratory biochemist Gregory Hannon, a former chair of the Integration Panel, says the program supported his work using RNAi libraries to screen for genes that allow breast tumor cells to survive. The strategy was too "risky" to have a shot at NCI funding, Hannon says. The program does have some detractors. Biostatistician Donald Berry, of the University of Texas MD Anderson Cancer Center in Houston, says that although he is "a big fan of Fran Visco and the NBCC" he thinks the DOD program would have benefited from input from a broader array of advocacy groups. The advocates on the Integration Panel tend to reflect Visco's and the coalition's views, he says. Others see nothing amiss with the coalition's impact and Visco's long tenure on the Integration Panel. Visco's vote is just one of about 15, and she "embodies the guiding principles behind the program," Hannon says. "They [NBCC] have a lot of influence because the plain absolute truth is, there would not be this program without them," Slamon says. If there is a shadow looming over the DOD breast cancer program, it is that each year there is a chance that Congress will decide to kill it. With many champions having left Congress or planning to soon, including Harkin, Visco and her colleagues are working to cultivate new backers. One factor in their favor is that Congress over the years has expanded the program into a much larger Army peer-reviewed effort targeted for a range of diseases, from prostate cancer to prion disease, giving it broad appeal. ## Agents of change The National Breast Cancer Coalition now wants something more radical. Some 20 years and$3 billion after the coalition's founding, "there hasn't been much in the way of real breakthroughs in breast cancer," Visco says. The incidence in the United States is still rising, and mortality is down only slightly, Visco argues. And there is too little research on prevention, she says. That led to the coalition's controversial new goal, announced in September 2010: to "end breast cancer by January 1, 2020." In an editorial on 29 November 2012, Nature called the 2020 goal "misguided" and wrote "[d]iscovery does not answer to deadlines."

Visco points out that the coalition has since emphasized that the specific goal is to "know how to end breast cancer by 2020." To do that, NBCC is encouraging research on two problems: how to prevent cancer with a vaccine and how to stop primary tumors from spreading, or metastasizing. These questions came from the advocates, not scientists, Visco says: "I think advocates can have a better idea than scientists because we're not just thinking about getting published or getting funded. We're thinking about what's best for women."

The coalition is using funding from a foundation to support seed grants for a few projects—chief among them genomics studies looking for antigens on breast cancer cells or signs of a virus that could be used to design a preventive vaccine. Many breast cancer researchers have dismissed preventive vaccines as a long shot. And that's exactly why the coalition should encourage this research, Visco says. We're trying "to be disruptive and to change things. So this is our way of bringing back urgency to breast cancer."