In which we hear of p53’s place at the cutting edge of gene therapy and personalised medicine, which are revolutionising the treatment of cancer – and, some predict, will remove the threat of ever dying of cancer from today’s young people.
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[p53] already, with no help from doctors, stops incipient cancer millions of times every day. Scientists do not have to top the elegant system that nature has engineered. They just have to harness it.
Sharon Begley
‘If you peer into cancer cells – and we’ve got amazing technologies now to catalogue all the things that are happening – and you look at them from an evolutionary point of view, it turns out that even within a single tumour there are many, many different subspecies of cells that have evolved in slightly different ways,’ says Gerard Evan, whose remarks about the rarity of cancer opened this book. ‘And here we are, basically trying to wipe out the entire tumour while keeping the patient alive at the same time. It’s a tough deal!’
But Evan is not disheartened. Indeed, as evidence mounts that cancer is an even more complex disease than anyone realised – a hotbed of evolution that makes of tumours a constantly moving target for therapy – he remains decidedly optimistic. Why? ‘Let me give you an analogy,’ he says. ‘Absent of cancer, human beings have been subject to terrible diseases of cells that grow inside them and invade and spread. These cells are genetically very heterogeneous, they exchange genetic information one with the other, and they grow like crazy . . . They’re called bacteria, right?
‘A hundred years ago you’d have looked at all the infectious diseases and said, “Oh my God, we’ve got to have a cure for each one – there’s TB in the lung, and in the bone, and then there’s this and that . . .” But it turns out they share a great deal of commonality, and if we hit them with antibiotics we can more or less eradicate, at least for a time, infectious disease due to bacteria. Now the fact is, bacteria are much, much more genetically complex and heterogeneous and hardy and resourceful in the evolutionary sense than cancer cells.’
The task for drug developers, Evan believes, is to find the commonality of cancer – the ‘mission critical’ mutation without which no tumour can survive. Not everyone agrees with this analysis; most cancer researchers are still backing the idea of targeted therapy tailored to the individual patient’s tumour characteristics. But whatever the perspective, tumour suppressors are obvious candidates for investigation, and much of the effort of academic researchers and their counterparts in the pharmaceutical industry is focused on repairing or enhancing the body’s natural capacity to single out and eliminate rogue cells.
People have high hopes and many imaginative ideas for p53-based therapies, though the journey from the lab to the patient’s bedside is often frustratingly slow. Ironically, as the explosive speed of technological advance makes it ever easier and quicker for scientists to develop potential new drugs, the rules and regulations governing the process get ever more tight: it typically takes a decade or more for a promising new therapy to be approved for use on patients. Very many prototypes never make it that far; drug development is, by its very nature, a painstaking process of trial and error, but even the ‘failures’ teach valuable lessons along the way.
VIRUSES AS DRUGS
The first person to try p53-based therapy in humans was Jack Roth, who in 1996 recruited to his study nine patients with inoperable lung cancer whose tumours were no longer responding to conventional therapy. In a pleasing twist to the story, Roth’s therapy made a virtue of the pernicious properties of viruses – the fact that the only way they can survive and reproduce is to invade the living cells of the host organism and hijack the machinery of replication. Using genetic engineering, he and his colleagues converted a common virus into a vehicle for transporting good copies of p53 into cells where the gene is dysfunctional. This engineered virus they injected direct into the patients’ tumours and found, to their gratification, that the strategy worked: the p53 gene was successfully transferred to the tumour cells; it switched on to produce healthy protein, and the patients suffered no significant side effects.
However, the viral vector, or delivery vehicle, proved poor at evading the sentries of the immune system, and in subsequent prototypes the scientists coated the virus with a substance to give it a better chance of killing tumour cells before being wiped out itself by the immune system. They also changed the delivery vehicle from a retrovirus to an adenovirus – the type that causes the common cold and other respiratory infections.
Therapies based on this design have been tested now in thousands of patients in clinical trials mostly in the US and China. They have proved effective, especially when used in conjunction with conventional chemo- or radiotherapy. Patients also need to be carefully selected for their suitability, since the treatment works better under some conditions than others. It tends to be most effective, for example, in tumour cells in which existing wild-type p53 protein is trapped by over-expression of its natural controller Mdm2; or when mutant p53 protein is produced at such low levels in the cancer cells that it cannot overwhelm the wild-type protein produced by the gene therapy. (You will remember that in some cases where a person has a mutant and a wild-type copy of the p53 gene, the mutant protein is powerful enough to knock out the function of the wild-type protein – the so-called ‘dominant-negative’ effect. Someone with such a powerful mutant will not be a good candidate for the gene-transfer therapy.)
Roth’s pioneering work in the mid-1990s led to the development of two trademarked products, Advexin in the US and Gendicine in China. As well as being injected directly into the tumour, these can be administered by injection into an artery or vein like chemotherapy, and have been tested in a number of tumour types including lung, liver, and head and neck with varying degrees of success. They seem to be effective also, used alone, in preventing early lesions in the mouth from turning malignant. In 2003, Gendicine was approved by the Chinese regulatory authorities for use in the clinic. The first gene-therapy product to receive official approval anywhere in the world, it is used today, in conjunction with radiation, to treat patients with head and neck cancer in China.
In 2007, Zhang Shanwen of Beijing Cancer Hospital, who chaired the clinical trials of Gendicine, gave an indication of its effectiveness. At a conference in China, he presented data from a trial in which 26 patients were treated with gene therapy plus radiation and 27 controls were given radiotherapy alone. Seventeen of the 26 patients who received the combined therapy were still alive five years later, of whom 16 remained completely tumour-free. Of the 27 controls given radiotherapy alone, 14 were still alive five years later, 10 of them tumour-free.
However, despite some remarkable individual success stories and despite being almost identical to Gendicine, the US product, Advexin, has had a rocky ride. The quest to get this product – considered an ‘orphan drug’ because of its limited market potential as a therapy primarily for head and neck cancer – into the clinic has been enormously expensive. When the US Food and Drug Administration (FDA) declined approval in September 2008 because there was not enough evidence of its effectiveness, the manufacturer, Introgen Therapeutics Inc. of Houston, went bankrupt. Today, Vivante, a small company that rose from the ashes of Introgen and was itself acquired in 2010 by the Swiss-based giant Lonza, holds the licence for Advexin and continues the quest for approval from regulatory authorities in the US and Europe. Meanwhile, the Chinese manufacturer of Gendicine is also seeking FDA approval for its product in the US and in India.
In the early 1990s, Frank McCormick at the University of California, San Francisco, began developing a therapy that uses the common-cold virus in a very different way. He had observed that cancer cells and adenoviruses share some important characteristics, one of which is that in order to stay alive they need p53 to be out of action. Here was a trait he could exploit. But it needed a good deal of engineering to ensure that the virus would target and kill only cancer cells and not cause more widespread infection. Essentially, McCormick removed the mechanism by which the virus itself normally knocks out p53 when it enters our bodies. This meant that his engineered virus could survive only in cells which already had no functioning p53 – that is, cancer cells. In these the virus grows and multiplies until the cells literally burst. However, if the engineered virus invades cells with functioning p53 – i.e. non-malignant cells – it withers and dies because it no longer has the machinery to knock out the tumour suppressor. The process by which the cancer cells burst is known as oncolysis, and part of the beauty of McCormick’s mechanism as a therapy is that engineered virus particles spilling from the burst cells can infect and destroy neighbouring cancer cells in the same way, but pose no threat to normal cells in the body.
In 1992 McCormick co-founded Onyx Pharmaceuticals Inc. to develop his idea, and in 1996 the therapeutic agent ONYX-015 entered clinical trials in the US – the first engineered oncolytic virus ever to be tested in humans. Those early trials, first on patients with head and neck cancer and then on those with a variety of other tumour types, looked good. The gene-therapy community was riding high. Then came a body blow.
In 1999, 18-year-old Jesse Gelsinger, who had enrolled voluntarily in a clinical trial at the University of Pennsylvania, died suddenly of multiple organ failure after his immune system over-reacted catastrophically to the agent he was given. The product under trial was an engineered adenovirus carrying a gene to correct the serious but rare liver disorder Gelsinger had been born with that leads to the build-up of ammonia in the bloodstream. After his death, the FDA temporarily suspended clinical trials of all gene therapy and subsequently tightened the rules on safety precautions. These were difficult times for pharmaceutical companies developing such agents, and in 2003 Onyx sold the licence for ONYX-015 to the Chinese company Shenzhen Si Biono Gene Technologies Ltd.
In the meantime, China itself had been developing an oncolytic agent very similar to ONYX-015. Oncorine, manufactured by Shanghai Sunway Biotech Ltd, was the first engineered oncolytic virus worldwide to reach the medicine cabinet when it was approved by the Chinese regulatory authorities in 2005. This approval raised the spirits of the depressed gene-therapy field. Today Oncorine is used in China in conjunction with chemotherapy (as an alternative to Gendicine) to treat tumours of the head and neck, and the data from trials suggest it is roughly twice as effective as chemotherapy alone. The goal of the Chinese companies is still to obtain approval for ONYX-015 or Oncorine for use in the US and Europe.
However, the propensity of the viral vector to be detected and wiped out by the patient’s immune system before it can deliver its cargo to the cancer cells remains a major challenge for scientists working to refine gene therapy. Another challenge is to find ways of reaching the scattered metastases with these drugs, for it is these secondary tumours that tend to kill the patient with cancer.
SMALL MOLECULES KICK-START STRESS RESPONSE
Other new strategies for treatment being explored start with the fact that in very many cancers p53 is not mutant, but the normal protein is inactivated by some other mechanism. In cervical cancer, for example, around 90 per cent of cases are caused by infection with human papilloma virus (HPV), a sexually transmitted disease that can also cause genital warts. Scientists at the US National Cancer Institute discovered in 1990 that one of the viral genes in cells infected with HPV produces a protein called E6 that completely stymies the action of p53. ‘What happens,’ explained Karen Vousden, one of the NCI team at the time, ‘is that E6 binds to p53 along with some other proteins. The end result is that the p53 protein is degraded very rapidly – it’s just broken up into little bits – so the cell never manages to make any p53 protein that’s functional. It’s as though there isn’t any p53 at all.’ Preventing infection with HPV in the first place was the obvious solution here, and a vaccine capable of doing just that was approved for the market in autumn 2005.
But the HPV story is an unusual one. More typically the normal p53 protein is prevented from carrying out its functions by abnormalities elsewhere in the tumour-suppression pathway, such as over-zealous behaviour on the part of its controller, Mdm2. This, you will remember from Chapter 13, is the gene switched on by p53 that produces a protein that, in its turn, binds to p53 protein and marks it up for destruction. This dance of death between p53 and Mdm2 goes on in an endless cycle, taking about 20 minutes to complete each time. In this way, Mdm2 ensures that the enormously powerful p53 – with its ability to kill cells or stop them dividing – is kept in check until needed. Once researchers began to understand this feedback mechanism, they figured that if they could release p53 from the clutches of Mdm2 by blocking the interaction between the two proteins, they should be able to reactivate normal p53 in cells where it was abnormally restrained – that is, cancer cells. In such cells, they reasoned, p53 is like a loaded gun primed to go off, but with the trigger jammed; the challenge was to find something that would release the trigger.
Working in his Dundee lab, David Lane became, in the late 1990s, the first person to manage to do this, with a tiny molecule that plugged the docking site between p53 and Mdm2. Because of the awkward shapes of proteins in general and the flexibility of p53 in particular, this was a huge technical challenge, Lane commented when I spoke to him for this book. ‘As tough as the search for antiretroviral drugs for HIV?’ I queried. ‘HIV is a very instructive example, actually,’ he replied. ‘When I was growing up as a young microbiologist, I was told there would never be a drug to treat a virus; it would be impossible, because the viruses are so close to the host and they use the host machinery. I was also told you’ll never get a drug that inhibits a protein-protein interaction. And the immune system will never have a role in helping to clear cancer cells. So you know, you get told these things with absolute certainty by people . . . And of course they’re always wrong!’ he laughed.
Today there are at least half-a-dozen drugs in development that disrupt the bond between p53 and Mdm2. The earliest and best known is Nutlin, produced by the pharmaceutical giant Hoffmann-La Roche since 2004 (the drug derives its name from the company’s research institute in Nutley, New Jersey, where it was developed). But the initial excitement generated by Roche’s success soon gave way to serious concern. The first reports of Nutlin described experiments with cells and tissues growing on gels in Petri dishes in the lab. But a mouse experiment reported in 2006 that uncoupled p53 from its controller had a catastrophic outcome that gave everyone working with this strategy pause for thought.
Having engineered a mouse with the gene for the p53 controller Mdm2 knocked out, scientists in Gerard Evan’s lab gave the animal a drug to switch on the tumour suppressor. With no controller, the p53 protein went into overdrive in cells throughout the body, resulting in mass, generalised apoptosis – effectively a mouse that melted. In fact, the mouse with no Mdm2 at all was not a relevant model for Nutlin and other such drugs, which are designed to uncouple p53 from its controller only transiently. However, it did raise questions about how to limit the destructive activity of p53 to the tumour sites. This is something drug developers have worked hard on, and modern versions of the drug show very little activity beyond their target cells.
In the lab, Nutlin has inhibited the growth of cells taken from a wide range of cancers, including colon, lung, breast, skin and blood, and it has shown activity in animal models too. But the results have been puzzlingly inconsistent, says Lane, who works closely with the Roche team on the continuing development of Nutlin. In recent experiments with acute myeloid leukaemia (AML), for example, they treated cancer cells from a number of different patients in Petri dishes; they found that while all the cells went into growth arrest at the same low dose of the drug and some committed suicide, it took 10 to 20 times the ‘normal’ dose to induce apoptosis in others. What was going on? No one is certain yet, though a reasonable hypothesis is that the balance between pro- and anti-suicide proteins active in a cell at the time of treatment affects its sensitivity. Until they understand fully the forces at work, the drug developers will be unable to say exactly which patients with AML should be treated with Nutlin and at what dosages to induce the desired effect, cell death, says Lane.
Meanwhile, researchers are investigating the use of Nutlin in combination with conventional chemo- and radiotherapy, and have found it to be more effective than using either Nutlin or conventional therapy alone in a number of tumour types. One objective of combination therapy is to harness the synergy between the different agents in order to be able to reduce the dosage of the conventional drugs – and thus their distressing side effects for patients – without reducing effectiveness. This is a pressing need in the case of sarcomas, which include bone cancers and are among the most common cancers in children, and here Nutlin looks promising. In 2011, scientists trying to kill sarcoma cells in the lab found they could reduce the amount of some conventional drugs by a factor of 10 when they used them in combination with Nutlin and still achieve the same or a better result as when they used the chemotherapy drugs alone.
Breaking the bond between p53 and its controller Mdm2 is such an attractive option for drug developers that a number of big international pharmaceutical companies, including Merck and Sanofi, and several smaller ones are in the race to get a drug of this kind into the clinic. Until they can do so, however, they still need definitive answers to the vital questions: what effect do drugs of this nature have, if any, in normal cells and exactly how toxic might they be?
Galina Selivanova at the Karolinska Institute in Stockholm is working on a drug of this design which she has named RITA. She points out that in order to kill cells, it is generally not enough for p53 simply to be present; to become active, the tumour suppressor needs to receive clear signals that the cell is under stress – signals that are likely to be strongest in cancer cells. ‘My hope is that if you have an Mdm2 inhibitor which is not too strong – maybe it’s enough to release just some p53 from Mdm2 – it will not have very drastic effects in normal cells. But in tumour cells, where you have all these signals which are activating p53, it will kill.’
When she left Russia in 1992 with a PhD in bacterial genetics from Moscow University, Selivanova intended to spend just three months of summer gaining experience with work on higher organisms before returning home. However, she joined the lab of Klas Wiman at the Karolinska Institute, discovered p53 and never went back. ‘It was so exciting from the start,’ she told me when I met her at a mutant p53 meeting in Toronto. ‘p53 is unbelievably interesting. Everything you do opens new questions, new perspectives.’ She joined the p53 community just as people were beginning to think seriously about translation – how they might use the wealth of knowledge they had accumulated to improve the treatment of people with cancer. It was a topic with personal significance: Selivanova had seen her own mother die of a brain tumour, and she soon found herself drawn into the quest.
Besides her own work with RITA, she and Wiman have worked together on another drug, known as PRIMA-1, that is turning out to be one of the most exciting p53-based therapies in development. The drug is designed to work in cancer cells where p53 is mutant and the protein it produces misshapen so that it cannot bind to DNA, as it should, in order to switch on other genes. PRIMA-1 is able to restore the mutant protein to its normal shape, and serendipity played a large part in its discovery. In 1995, the two scientists were studying small scraps of protein called peptides, looking for ones that could regulate the activity of p53. They were intrigued to discover one peptide that was able to activate both normal and mutant p53.
This was clear evidence that ‘mending’ mutant p53 was possible, and Selivanova was very excited. ‘It was fantastic,’ she recalled with a smile. ‘I wanted, of course, to go out and cure tumours – at least in mice!’ But the peptide proved unworkable as a drug: in a living organism these scraps of protein are poor at entering cells and are quickly broken down and recycled. What was needed was a chemical compound, a small molecule that would perform the same tricks.
Working with Wiman and a new postdoc, Vladimir Bykov, Selivanova screened thousands of compounds from a library of possible candidates provided by the US National Cancer Institute. In 1999, the three discovered a molecule they named PRIMA, an acronym for ‘p53 reactivation and induction of massive apoptosis’ that appealed to the scientists because it also implies something that is first class. Experimenting with the molecule they found, to their gratification, that it is effective with a wide range of p53 mutations, and therefore potentially useful in treating many different tumour types. They published their results soon afterwards, ‘and PRIMA attracted a lot of media attention,’ Wiman, a tall, soft-spoken Swede, told me when I visited him at the Karolinska Institute. ‘I was on TV and in newspapers and journals around the world, because the concept of having a small molecule that will make the cancer cells commit suicide is so appealing.’
So how does PRIMA-1 work? Wiman and Bykov discovered, to their surprise, that both PRIMA-1 and a very similar compound known as PRIMA-1 MET are converted to another compound that binds tightly to p53 protein and refolds it. ‘This was a very important and exciting finding since it gave us a better understanding of how these compounds can reactivate mutant p53,’ said Wiman.
In partnership with the Karolinska, he, Bykov and Selivanova set up a small biotech company to develop PRIMA-1 MET for the market. It has been a steep learning curve. ‘As scientists you need to work with company people – a completely different culture,’ commented Wiman. ‘Suddenly there are people in suits, board meetings, talk about money . . . And then you interact with clinicians too. So there are three worlds and you all have to work together all the way through. We had no idea what was involved when we started.’
The company has taken PRIMA-1 MET through a phase 1 clinical trial, which involved 22 patients with cancer of the prostate and blood being given a short course of the drug by injection. Phase 1 trials are designed to test patients’ tolerance to a potential new drug, and to find out how it disperses in the body and how long it persists. The results, published in 2012, were promising: they showed that PRIMA-1 MET is not toxic and that side effects – including dizziness and fatigue – are mild.
Phase 2 clinical trials, designed to prove that the drug works in people as it does in the lab and in animal models, are the next step. The Karolinska researchers and their company are hoping to test PRIMA-1 MET in combination with conventional chemotherapy in cancer patients, where the two drugs are expected to act in synergy: while PRIMA-1 MET restores mutant p53 to its normal shape and function, the other drug will cause DNA damage that sends clear signals of stress to trigger apoptosis. But this is where the hurdles en route to the clinic really begin: staging a phase 2 trial for PRIMA-1 MET is likely to cost millions of euros, said Wiman. A tiny biotech company like theirs needs to find a partner with serious money to invest.
A doctor in Denmark who has seen the effects of PRIMA-1 MET on lung-cancer cells in the lab and in mice is so excited by the drug that he has offered to set up a trial himself. The MD Anderson Center, too, is keen to run a phase 2 trial of PRIMA-1 with cancer patients. But finding the funds for all these activities remains a huge challenge, and so far Big Pharma has shown little interest in small molecules that restore normal shape and function to mutant p53 because it is still not entirely clear how they work.
SMART THERAPY
A problem that dogs the field of cancer therapy is the issue of drug resistance. The extreme instability of cancer cells and the terrible speed with which they pick up mutations mean that they are likely to find a way round a targeted drug before too long, no matter how clever the design, as the cells that survive the initial onslaught of treatment give rise to equally hardy clones that grow into resistant tumours. To minimise the prospect of failure, oncologists typically treat their patients with a combination of therapies – either a cocktail of drugs, or a drug together with radiotherapy. With this strategy, cancer cells that are not affected by one drug should be hit by the other.
Researchers are also investigating the use of drug combinations in a novel kind of p53-based treatment called cyclotherapy. One of the biggest shortcomings of conventional chemotherapy, which is ‘cytotoxic’ (meaning that it’s a cell poison) and targets the body’s rapidly dividing cells, is that it is indiscriminate. Cancer cells are by definition fast-dividing, but so too are the cells in the hair follicles, lining of the gut and bone marrow, which sustain collateral damage during chemotherapy. But hair loss, nausea, diarrhoea, anaemia and depletion of the immune system are not just distressing side effects for the patient, they are potentially deadly and they limit the dose of cytotoxic drugs the oncologist can administer to attack the cancer.
The principle behind cyclotherapy is that patients be given one drug to ‘protect’ the healthy cells from the chemotherapy by temporarily stopping them from dividing, while their cancer cells (which continue to divide and therefore remain targets of the chemotherapy) are blasted with a second, cytoxic drug given simultaneously. With healthy cells protected, the theory goes, the oncologist will be able to increase the dose of the cytotoxic drug and thus maximise its potential to wipe out the tumour. But even if it falls short of clearing the cancer completely, cyclotherapy will make chemo a lot less unpleasant for the patient because it will limit the side effects by sparing the cells of the hair, gut, bone marrow, etc. from the full force of treatment.
In laboratory tests, Nutlin is looking the most promising of a number of similar drugs used to protect the healthy cells. However, cyclotherapy is still a few years from the clinic. Researchers still need to work out which combinations of drugs work best, with what tumour types and in what quantities. The arrest of healthy cells mid-cycle must be reversible: too high a dose of the protective drug, for example, could cause healthy cells to senesce, but too low a dose might not arrest them for long enough to protect them from the cytotoxic drug. And no one is sure yet how well cyclotherapy works in living organisms: as of 2012 there was only one published report of an experiment in mice. However, one of the main constraints on cyclotherapy is the fact that neither Nutlin nor any of the other potential ‘protectors’ has yet been approved for use in the clinic in its own right.
NEW LIGHT ON OLD TREATMENT
Despite the frustratingly slow progress of brand new p53-based therapies, scientists’ understanding of p53 is already beginning to have an impact on the treatment of cancer patients: it enables oncologists to make more rational decisions about the use of conventional chemo- and radiotherapy.
Chemotherapy has a colourful, if unfortunate, history. Its origins go back to World War I, when the Germans used mustard gas in the trenches of Europe to devastating effect. The use of chemical weapons was banned by the Geneva Protocol of 1925, but not the possession of such weapons, and the Americans continued to develop and stockpile them. In December 1943, a US cargo ship, the SS Harvey, secretly carrying mustard-gas bombs to the Mediterranean war front, was sunk in a German raid on the port of Bari, southern Italy, and a cloud of gas drifted over the city. No one knows how many civilians were affected, but more than 600 military personnel were hospitalised and 83 died. During autopsies of the victims, pathologists found evidence that the normally fast-dividing cells of the bone marrow and lymphoid tissues had been suppressed. From this observation came the idea that perhaps such an agent could be used to attack the rapidly dividing cells of cancer.
Soon scientists were doing experiments with mustard gas in mice. Encouraged by the results, they moved cautiously on to testing the agent in humans. The first human subject was a patient with lymphoma – cancer of the lymphoid tissue – and his doctors observed with delight the dramatic shrinkage of his tumours after administration of the drug. Unfortunately the effect was short-lived, but it galvanised the cancer community: here at last was a new way of treating the disease. For many centuries, surgery had been the only option for getting rid of tumours, and patients’ long-term survival chances were minimal.
Over the decades since, many different cytotoxic drugs have been developed – all on the same principle, that they are poisonous to cells. But while chemotherapy has been found to work wonderfully well in some tumours, it does not work at all in others. And in some it works for a while and then stops. Why is the response so varied? Until p53 research began offering clues, no one had an answer. Today we know that both chemo- and radiotherapy work not by killing cancer cells directly, in a sledgehammer kind of way as had been assumed, but much more subtly: typically, these therapies work by inducing cancer cells to commit suicide in response to damage of their DNA – the normal response to cell stress, mediated by p53.
Scott Lowe, whom we met in Chapter 12 creating mouse models and making groundbreaking discoveries about apoptosis and p53, was one of the first to recognise the tumour suppressor’s central role in conventional therapy. To recap, Lowe subjected the highly sensitive thymus glands of his mice to radiation and discovered that the cells with normal, functioning p53 died very quickly by apoptosis, but cells with mutant or no p53 were resistant to radiation and survived. Confirming p53’s role in apoptosis in response to radiation set Lowe wondering more generally: could p53 be responsible for the effect of radiation – and perhaps cytotoxic drugs also – in cancer therapy? As an idea, it was incredibly simple, and so obvious in retrospect, but revolutionary at the time.
‘Here was a situation where the hypothesis was that if p53 is mutant, the standard chemotherapy drugs are less likely to work,’ explained Lowe. ‘In the case of leukaemias and lymphomas what we would have predicted holds true. But now, 17 years of subsequent research says of course it’s more complicated than that.’
In leukaemia and lymphoma cells, p53 is almost always normal and, as one would expect, these cancers are highly sensitive to chemo- and radiotherapy. But in solid tumours (cancers of the organs rather than the blood), the picture is much less predictable – and sometimes it is counter-intuitive. In some types of cancer, cells with mutant p53 are more responsive to cytotoxic drugs than are cells with normal p53. This is the case with glioblastoma, an aggressive tumour of the brain, for example. So what is going on?
One explanation is that in these cases, the cells with mutant p53 are indeed killed in the sledgehammer way oncologists originally imagined. They sustain severe damage to their DNA that cannot be repaired because p53 is out of action, nor can cell division be arrested. The cells carry on chaotically through the cycle and eventually succumb to what is called ‘mitotic catastrophe’ – wholesale failure of the machinery of replication. This scenario implies that it is essential for oncologists to know which way a tumour type will react to conventional therapy, depending on its p53 status. But things can get even more complicated.
In some cases, giving chemo- or radiotherapy to patients whose cancers have normal p53 can actually make things worse. Cell death, as we know, is just one of several options chosen by p53 in response to damaged DNA. It can also choose to arrest the cell mid-cycle and send in the repair team before releasing the cell to carry on replication. Or it can condemn the cell to senescence – permanent arrest, which we know from the chapter on ageing can eventually stimulate cancer in neighbouring cells. Thus cancer cells that are not killed by chemo- or radiotherapy can be the seed stock for further tumours – and sometimes these new tumours are especially aggressive simply because the cells are survivors of highly toxic treatment, and bred for resistance.
This makes sense, but it is only a hypothesis at present – there are no experimental data to prove it definitively. One source of confusion is the fact that, in the vast wealth of research that is carried out on p53, there is so little consistency in the methodology that it is hard to compare results. ‘In experimental systems we have all kinds of effects,’ said Pierre Hainaut. ‘You can always get an experimental system to behave as you would like it to, as an investigator! Now if you go to real life . . .’ Hainaut sat me down in front of the computer in his study at his Lyon home and brought up a paper he was about to submit. It was an analysis of a number of clinical trials involving the use of a common chemotherapy drug, Cisplatin, in lung-cancer cases. Overall, the effect of the drug was small, but he and his colleagues wanted to know whether the p53 status of an individual patient’s tumour influenced the outcome of Cisplatin treatment. For their analysis they had before them the biggest data set of its kind. It contained information about the outcome of treatment, plus the p53 status of the tumours, for 1,200 cancer patients from four trials, conducted in Canada, the US and Europe.
The researchers found – unsurprisingly – that patients whose tumours had normal p53 did a lot better than those with mutant p53. But what did surprise them was that patients with some specific mutants – but, crucially, not others – got dramatically worse after Cisplatin treatment. Their tumours spread aggressively and many patients died even more quickly than they would likely have done with no treatment at all. Hainaut was not certain, at that point, whether it was the metastases that killed the people – he was awaiting further information from a statistician – but that was his hunch.
Whatever the final cause of death turns out to be, knowing the p53 status of lung tumours will be useful in deciding who should receive Cisplatin therapy and who should not. ‘We are not doing well with lung cancer,’ Hainaut reflected as he scrolled through his paper on the computer screen. ‘There are probably 1.5 million people in the world with this type of cancer. Maybe 500,000 receive this treatment every year – and they receive it “blind”, because p53 is not being tested by mutation in these patients up front. Such a test would clearly improve the outcome. It would be really worthwhile . . . That’s the lesson of our analysis.’
The situation Hainaut was describing was specific to lung cancer, with certain p53 mutations, treated with Cisplatin. But the lesson holds true more generally. What scientists have discovered about p53 and its role in conventional therapy offers cancer specialists a tool for making more rational decisions about how best to treat their patients. This is especially true when p53 status is part of a wider analysis of the genetic make-up of a tumour, because so many things besides this tumour suppressor have an impact on treatment. At present such tests are rarely offered in cancer clinics, but things are changing fast. As full genome sequencing becomes ever easier, quicker and cheaper to perform – and as the new gene therapies that target the defects specific to an individual patient’s tumour begin to reach the clinic – genetic analysis will become a routine part of diagnosis and treatment. Genetic analysis is an essential part, too, of the latest strategies for cancer prevention.
THE BEST CHANCE OF SURVIVAL
Compared to new treatment ideas, prevention studies have a tough time attracting cancer-research funds. The science of prevention is not as sexy; it doesn’t offer the same rewards to Big Pharma; and besides, it’s easier to get excited about tumours that are cured than about tumours that just don’t happen.
Nevertheless, Bert Vogelstein is not deterred. ‘We believe the major impact on cancer over the next half-century will come not from treating advanced cancers, but from preventing cancer – in particular from detecting tumours at a very early stage,’ he said. ‘Virtually all cancers are treatable by surgery, without the need for any chemotherapy or radiation, if they’re caught early enough. That’s definitely true for colon, but it’s also true for many other tumours. It’s an underlying principle.’
For a number of years now, Vogelstein’s lab at Johns Hopkins has been busily engaged in developing tools to look for evidence of early cancers. They are focusing their efforts on detecting biomarkers – bits of mutant DNA sloughed off by cancer cells that might be swilling around in a sea of normal DNA molecules in the blood, urine, stools or sputum, bearing witness to the presence of furtive disease. ‘The best marker, the best gene, is obviously p53, because it’s mutant in more tumours than any other gene – that’s the bedrock of this test,’ explained Vogelstein.
The body fluid in which a biomarker is found is often a good indicator of where the tumour is developing: urine suggests bladder cancer, for example, stool suggests colon cancer and sputum suggests lung. By late 2012, Vogelstein’s team had investigated more than 700 cancers, starting with advanced tumours, to see if they could find free-floating biomarkers. ‘In advanced cancers of most tumour types – that is breast, colon, pancreas, lung – you can detect well over 90 per cent of them in the blood,’ he commented. For advanced colon cancer the researchers’ detection rate in stool samples is close to 100 per cent, and even in relatively early, pre-metastatic cancers it is 85–90 per cent. ‘This test is starting to rival colonoscopy in sensitivity,’ said Vogelstein. He reckons that even in blood samples, his team has more than a 50/50 chance of detecting colon cancer before it has spread. ‘And if you can detect even 50 per cent of cancers at a stage when they’re curable that would be massive.’
Researchers working on the problem of liver cancer in West Africa have found biomarkers in the blood that can be used to screen for the disease before symptoms arise. In this region, you will recall, liver cancer is often associated with aflatoxin contamination of food crops, and DNA molecules released into the blood from a diseased liver show the characteristic fingerprint mutations in the p53 gene. Elsewhere, too, scientists are exploring the possibilities of using the presence of mutant p53 in body fluids to screen for early cancers.
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Such screens have still to be widely validated and refined before they reach the clinic. However, many scientists working on the front line of p53 believe we are on the threshold of a golden age in cancer prevention and cure. In coming years we should expect to see:
• gene therapy become routine treatment for cancer as researchers perfect the technique for modifying viruses as delivery vehicles (dozens of people with diverse genetic disorders have already been treated successfully);
• more widespread use of genetic analysis of tumours, and the status of p53 being used to determine the best course of treatment and to predict outcomes and long-term prognosis;
• a dramatic decrease in side effects of cancer therapy as treatment becomes more accurately and exclusively targeted at tumour cells, and as strategies such as cyclotherapy are used to protect the body’s normal cells;
• a variety of p53-based drugs for use in different circumstances that are able to manipulate the tumour-suppressor pathway to kill cancer cells.
‘I’m very, very optimistic,’ said Gerard Evan. ‘I think we’re going to see dramatic shifts in our ability to treat and contain human cancers over the next 10, 15, 20 years.’ And he added, perhaps provocatively, ‘My daughter is 22 and my son is 21, and I can pretty confidently say they will never, ever have to worry about dying from cancer.’