p53

by Sue Armstrong

  A C Camargo, the largest specialist cancer centre in Latin America, was purpose built in 1953 on the site of a former Japanese temple a short distance from the busy boulevards of the city centre. This is the district of Liberdade, a network of narrow, shabby streets lined with small shops, businesses and open-fronted diners arranged on hills that afford sudden views across São Paulo, out on to green squares and old churches and beetling traffic. Liberdade is home to the biggest community of Japanese people outside Japan. With its complex of modern research laboratories and training facilities alongside the imposing white building of the hospital itself, A C Camargo dominates the district. Every year some 15,000 new patients seek diagnosis and treatment here. Around one in 20 of them comes from outside Brazil, attracted by the fact that A C Camargo’s oncologists are specialists in many different aspects of cancer and are highly skilled.

  Achatz is an oncogeneticist, a relatively young specialism in medicine that is concerned with cancer cases in which the faulty genes are inherited rather than occurring by chance during the course of life. She grew up in a cosmopolitan family in Rio de Janeiro, the daughter of an economist father and housewife mother, and the youngest of six children. On finishing high school she went to university in Paris to study art and design. But her plans for a career in the arts came unstuck when she joined a bunch of her university friends on a visit to India. The group spent time on the Kashmir border, camping in a desert area close to the site of a leprosy colony where the people were living as outcasts in caves in a stony hillside. Their plight, and their extraordinary resourcefulness, made a deep impression on Achatz. But it was a chance meeting with Mother Teresa, whose religious order ran the colony, that made her decide on a career in medicine instead. ‘It was an amazing encounter and I thought, well, I just have to go back and do something more worthwhile.’

  Achatz returned to Brazil and eventually to medical school in São Paulo – a training that was interrupted briefly by the birth of two of her children. Family life has always been important to her and she says, ‘I decided to go into genetics because I wanted to work with families. Mostly I wanted to work with cancer genetics, which was an emerging field.’ A C Camargo has one of the busiest oncogenetics programmes in the world, and during her first year there, while she was still a trainee, she saw 30 families with what she believed was LFS.

  ‘It really struck me because this was considered to be a terribly rare syndrome. There were only 280 families described in the world literature at that time – and I had 30. So I thought, either I’m over-diagnosing or something unique is happening here,’ she told me as we sat talking in her small, windowless office behind the lab in one of the research buildings. Maria Isabel is tall, slim and effortlessly elegant, with long brown hair which she occasionally ties back into a swinging ponytail. Dressed casually in white shirt and slacks, she sat behind her desk, always prepared for a phone call from one of her patients or a colleague or student.

  There are more than 70 cancer-predisposition syndromes, she told me, but she thinks she has an eye for spotting LFS, having seen her first family with the syndrome while she was still a medical student at another hospital in São Paulo. ‘This patient came just for clinical follow-up, but after the consultation she said, “Well, actually I am a cancer survivor. I’ve already had five different cancers.” I said, “You mean metastases?” And she said, “No, no, five different cancers.” Then she listed all the different tumours she’d had and she said, “This is something really common in my family – we have so many cancers . . . It just happens, and we get better afterwards.”

  ‘I was so fascinated that I studied and studied, and when I came to this hospital as a trainee, again, the first patient I saw was a Li-Fraumeni patient. They just kept coming and coming, till I had by the end of the year these 30 families with LFS.’

  One of the ways scientists communicate their findings to each other is through the medium of posters – short summaries of their research projects that they are invited to pin up on special display boards in side rooms of scientific meetings. In spare moments, delegates can wander around reading the posters at their leisure. Achatz’s boss at A C Camargo encouraged her to submit a poster on her LFS families to a cancer conference scheduled for France in the winter of 2002, and when it was accepted she and he flew together to Paris to attend. Achatz recounted how she found herself standing before her poster discussing her work with a tall, earnest, bespectacled scientist whose name she didn’t know, when an agitated official came to tell him he was due on stage any minute as chairman of the next session. As he hurried away, Pierre Hainaut handed her his card, ‘and I realised I had been talking to one of the top scientists of WHO’s centre at IARC,’ she laughed.

  As keeper of the p53 mutation database, Hainaut was well aware of LFS and its supposed rarity, and he was fascinated by Achatz’s story. He invited her to visit him at his lab in Lyon before flying home from the conference, and after much negotiation the two agreed to collaborate on research into the Brazilian phenomenon. Maria Isabel had never seen herself as a research scientist – ‘I was very happy being a medical doctor, taking care of my patients and trying to detect their tumours early,’ she told me. Besides, at the time of the Paris conference, she was pregnant with her fourth child and fully intended to drop back into the carefully constructed routine of family and hospital life she had left. Encouraged by Hainaut, however, she did a PhD and today she combines research, and the supervision of a bunch of students, with a busy medical practice at A C Camargo.

  Oncogenetics is a particularly demanding specialism, she told me, ‘because I bring my patients the bad news that their families will have to take special care of themselves forever. I’m not someone who performs surgery and heals people. I’m only able to offer a hand and walk together with them; I’ll never be able to withdraw their suffering.’

  She tells the story of three siblings, a girl and two boys, all young adults, who decided to join her research programme having watched their mother cope for years with multiple episodes of cancer. After counselling the three and making sure they understood the implications, Achatz took blood samples and sent them off for analysis. She was startled to learn from the mother the following week that one of her sons, a young man with a PhD in engineering and a good job, had left the clinic after the consultation and, without waiting for his results, quit his job, left his wife and two children, and had a vasectomy. He wanted to live life to the full while he had the chance, he said.

  ‘The thing is that the results came back soon afterwards and he wasn’t even a carrier,’ said Achatz, shaking her head at the needless tragedy of the story. ‘A very educated person . . . We took all the time we needed to counsel him. He understood it well. But this shows how big is the impact of having a genetically inherited disease: your education, your IQ, your background have nothing to do with how you react. Getting the information is just such a hit that no one can tell how you’ll take it.’

  At their first meeting in Lyon back in 2002, Hainaut asked Achatz to collect blood samples from her cancer patients in São Paulo and bring them to IARC for analysis. After extracting the DNA, the two scientists started by isolating and sequencing the sections of p53 in which the great majority of significant mutations – including the ones most commonly associated with LFS – occur. But out of 45 supposed LFS patients from whom Achatz had drawn blood, they found only three with mutations. Achatz was mortified. ‘I said, “Well, I was wrong. I over-diagnosed. I don’t have anything unusual here in Brazil.”’ But Hainaut wasn’t so easily put off. They must now sequence the whole gene, he said, including the sections outside the usual hot spots, at either end of the gene. Sure enough, they found that a good proportion of the individuals carried mutant p53, with a mutation at codon 337 being most common.

  Codon 337 falls in a part of the gene responsible for how the protein folds, and therefore how it interacts with other proteins in the cell. Hainaut was aware that this same, unusual mutation had been descr
ibed just the previous year, 2001, in association with adrenocortical cancer (ACC) – significantly also by scientists working in Brazil. ACC, you will recall, is the rare childhood cancer that Li and Fraumeni had investigated when searching for evidence of a cancer syndrome that runs in families. But the Brazilian scientists, led by Raul Ribeiro, claimed there was no evidence that the 337 mutation predisposed to anything other than ACC in children. This was not part of a wider cancer syndrome, they said, and in their paper for PNAS they gave a convincing explanation of why the risk should be limited to the one organ: that 337 was a ‘conditional mutant’ that caused the protein to misfold only at a certain pH level (a measure of acidity in the cells), a level found in the adrenal glands. They suggested, moreover, that an agricultural pesticide widely used in southern Brazil might be responsible for causing the mutation in the sperm or egg cells that resulted in children being born with faulty p53.

  ‘This was a story that kept coming up,’ said Hainaut, ‘and in my first contact with Maria Isabel we did discuss the possibility of the pesticide.’ Anxious to investigate further the connection between the 337 mutation and a wider spectrum of tumours than Ribeiro’s group had found, he flew to São Paulo to do further work with Achatz in 2005. She had recently begun collaborating with Patricia Prolla, who runs a medical genetics clinic at the big general Hospital de Clinicas de Porto Alegre, in the south of the country, and who was also seeing patients with apparent LFS. Together the three scientists travelled to Ibiuna, a small town on the outskirts of São Paulo where one of Achatz’s most open and accepting patients had promised to set up a family meeting at her home. The three found 26 members of the family crowded into the elderly matriarch’s kitchen, and over cakes and coffee round the kitchen table they listened to their stories the whole afternoon.

  ‘They’d seen so many deaths in their families and nobody had ever said there was something to be done, so they just thought, “Well, that’s our family,”’ Achatz told me. ‘Many of them spoke about being cursed.’ But the information the three scientists gave the men and women gathered there – representing three generations of the family – about LFS and the screening procedure that could determine who among them was a carrier of the mutant gene and who was not, kindled a spark of hope that challenged their fatalism. Achatz, Hainaut and Prolla finally left the house with blood samples from almost everyone in attendance.

  This was just a small grouping of the large extended family that lives in and around Ibiuna, however. Word of the meeting with the A C Camargo team soon spread among those who had been reluctant to delve too deeply into their painful history, and many more decided they wanted, after all, to hear why they were so cancer-prone and what could be done about it. A few weeks later, around 85 members of the family travelled to A C Camargo for a meeting with Achatz. ‘That was in 2005, and I still see them almost every week – one of the families comes here,’ she commented. ‘One cousin brings another cousin – they just keep coming.’

  WHERE DID IT START? HOW FAR HAS IT SPREAD?

  As the extent of the problem in the Ibiuna family became apparent, Achatz and Hainaut began to ask the questions: where did the mutation originate? And how far back did it go? With the help of a local priest who had access to parish records of marriages, births, baptisms and death, the researchers were able to trace the family back to the early 19th century and to draw up a family tree with several hundred people across eight generations. Wherever possible they added details about who had suffered from cancer and of what organs. The trail of disease goes back a long way and one of the family members suggested it could be part of their tropeiro heritage.

  The tropeiros were travelling traders, muleteers who supplied the early Portuguese settlers in the mining and farming communities dotted around southern Brazil with all manner of goods, as well as carrying mail and news from the outside world. The traders travelled great distances, were on the road for long periods of time and likely had girlfriends all along the route. In a scenario reminiscent of the AIDS pandemic, someone with a mutant gene could have passed it on in much the same way as HIV was disseminated along the trucking routes of Africa and Asia. Such a genetic fault is known as a ‘founder mutation’ – one that is introduced to a population by a single immigrant and can be traced back to an individual, a common ancestor, through the DNA. It was an idea that begged to be explored because, like a stone thrown into a pond, the ripples from a founder mutation can spread far and wide. How far had 337 mutant p53 spread in Brazil?

  It has turned out to be remarkably widespread. When Prolla and Hainaut did some research in Porto Alegre – also on the trade route of the tropeiros and their mules – to investigate the prevalence of the mutation in the general population, they came up with startling results. Prolla’s particular interest is breast cancer, and she was involved in a study of prevention strategies that recruited several thousand healthy volunteers from poor suburbs of Porto Alegre to test mammography. She and Hainaut secured permission to test the blood of 750 of the women. It was a shot in the dark; they had no idea what to expect, but they found two samples positive for the 337 mutation. ‘The frequency was significantly higher than we expected,’ said Prolla. ‘In studies that have been published in Europe and the US, the frequency of a germline mutation in p53 is about one in 5,000 individuals in the general population. If we really think that the frequency of the 337 mutant is somewhere around one in 300 or 400 in the south and southeastern regions of Brazil, for one single mutation, this is much more common – at least 10 times more common – than any other germline p53 mutation anywhere else in the world.’

  The extremely high prevalence was confirmed subsequently by a screening programme of newborn babies in the southern Brazilian state of Paraná. Of 171,649 babies whose parents agreed to the test, 0.27 per cent were found to be carrying p53 with a mutation at codon 337. Such findings were disconcerting: could this mutation really be a cause of the cancers, or was it just a red herring – a harmless variant of the gene – the scientists wondered? The evidence that the mutation is indeed harmful was strong, however. Besides the fact that the risk of ACC in carriers is 10–15 times higher than ‘normal’, women with the mutation tend to develop breast cancer at least 10 years earlier than those without.

  The ‘common ancestor’ hypothesis was confirmed when the scientists found that every LFS family member who had tested positive for the 337 mutant in Brazil had an identical version of p53 – there was no variation to suggest that the same mutation had occurred spontaneously and independently a number of times. This remains true for more recently identified cases also. The lack of variation tells us that the original case was relatively recent, said Hainaut. ‘It’s not an old founder effect that goes back a thousand years or so, or you would expect to see some kind of “drift” in the non-coding region of the gene.’

  There’s a postscript to the story of Prolla and Hainaut’s prevalence study in Porto Alegre. Prolla followed up their research by calling in the two women who had proved positive to discuss their family histories. Cancer was all too familiar to them, the women reported. And to everyone’s surprise they discovered, while drawing up family trees with Prolla to track the disease across the generations, that they were related: both reported the same distant cousin who had cancer.

  Not long after these consultations, the baby niece of one of the women developed ACC. Because of the awareness of symptoms raised by Prolla’s work with the family, the baby was brought promptly to her clinic, when her tumour was easily treatable. But her parents were reluctant to consent to surgery because of their religious beliefs – they were Jehovah’s Witnesses, whose faith prohibits blood transfusions. Much to their relief, the surgeons removed responsibility from their shoulders by securing a court order for treatment to save the child’s life, and in the event no transfusion was needed. Today she is a healthy little girl who visits Prolla’s clinic and the hospital regularly, along with her father and aunt, for her routine screening, as all three are carriers of the
p53 mutation.

  A MORE COMPLICATED STORY

  The tropeiro connection is intellectually appealing, and the Ibiuna family can trace their roots back to an ancestor, an immigrant from Portugal, who made his money as a travelling trader and bought land in the Ibiuna area to grow grapes for wine in the early 1800s. But in the end it can never be proven that he was responsible, and Hainaut felt from the beginning that the true story was probably more complicated. He has another hypothesis about the origins of the founder mutation that he feels is equally plausible and as a result has spent nearly two years investigating alternative possibilities, often accompanied by Achatz, as time permitted. The mutation is known to be concentrated in southern regions of Brazil, and the trail has taken the two to towns and settlements along the old trading routes between São Paulo city and Porto Alegre, 860km (530 miles) away in the state of Rio Grande do Sul.

  Codon 337 is a vulnerable site on the gene for mutations, explained Hainaut, and to investigate possible carcinogens he and Achatz travelled to a grim industrial town where the population is exposed to pollution from heavy metals, sulphur and other chemicals seeping from dirty mine dumps. But they found nothing of particular interest there. They visited a coastal town which was a popular entry point to the country for European immigrants in centuries past, and frequented by sailors from round the world, but again their amateur sleuthing turned up nothing.

  The third idea they followed took them to Araranguá, 210km (130 miles) north of Porto Alegre, which had been settled by the Portuguese since the early 1700s. They spent hours poring over the old archives in the town hall and here they felt they might be on to something. The records showed that the roads used by the tropeiros had started from Laguna, another small city some 100km (60 miles) to the north. They had been built originally on the orders of King João V of Portugal as part of his strategy to secure the territory inland, between the coast and the central plateau, for his crown. At the time, the area was occupied by indigenous peoples, with occasional incursions by Spanish troops from the Río de la Plata to the south – what are today the countries of Argentina and Uruguay. The king’s desire to control the area was driven largely by the fact that rich seams of gold were being mined further north, and he wanted to occupy this untamed land in order to prevent the Spanish from moving in from the south and seizing the mines. The people of Laguna were therefore recruited to build a route to carry soldiers and settlers as fast as possible towards the escarpment.