DNA detectives chasing the causes of cancer

But even after decades of investigation, we still do not know what's causing these hot spots.

East Africa is not the only place in the world where this happens. The Golestan region in Iran has one of the highest rates in the world and there are pockets of the disease in places as diverse as Henan Province in north-central China and southern Brazil .

Other parts of the world have their own cancer problems: there are strangely high rates of bowel cancer in Slovakia and Denmark, although they have low rates of liver cancer . People in the Czech Republic are more likely to have kidney or pancreatic cancer than the neighboring Austria and Poland populations.

Do these differences lie in inherited genetic variations or is it related to lifestyle? Is there an unknown carcinogen hiding in the environment? Or maybe it's a bit of the three? The wild differences in cancer rates around the world are a mystery – but a team of detectives is on the case.

By peering deeply into the DNA of cancer cells, Stratton and his team are looking for unique mutant signatures that different carcinogens and agents have left behind.

"I was interested in the idea that you can detect cancer exposures that have lasted 20 or 30 years," says Stratton. "A mutational signature is simply the pattern of mutations that is left behind by a mutation process, and a mutation process can range from the exposure of a cell to ultraviolet light to tobacco smoke or from endogenous processes. "

The Mutographs team recruits 5,000 people on five continents with five different cancer types, extracting and analyzing the DNA of thousands of tumors to build a massive database of mutational signatures – a little match causes to cancers worldwide.

The genetic fingerprint of cancer

At its heart, cancer is a disease of DNA. The human genome contains about 20,000 genes – the biological instructions that tell our cells when to grow and multiply, what work to do in the body, and even when to die – coded in long strands of DNA called chromosomes.

The DNA itself is made from four chemical building blocks, or bases, that are assembled in infinitely varied combinations. It is the order of these bases – adenine (A), thymine (T), guanine (G) and cytosine (C) – which transmits information in a gene, acting effectively as an alphabet molecular describing the recipes of life. Any modification of the letters of an important gene – for example, one that causes the proliferation of cells – can cause a multiplication of out-of-control cells.

Other modifications in other vital genes, as well as a cellular environment that allows or even encourages uncontrolled growth, will eventually lead to a tumor. If you can detect the mutations in the DNA that led to the development of a person's cancer and determine what caused them, then you should find the solution to their biological bias. But to do this, you must be able to read the DNA.

In the late 1970s, biochemist Fred Sanger developed a reliable method for reading the sequence of letters from a piece of DNA, and the Cambridge Institute bears his name as evidence of this groundbreaking discovery. Sanger's original sequencing technique was long and tedious, allowing scientists to read a few hundred basics at best. Thus, rather than looking at the six billion letters of the human genome in search of carcinogenic changes, the researchers began by focusing on a single gene, p53, which is defective in the majority of human cancers.

In the 1990s, Curtis Harris of the US National Cancer Institute and Bert Vogelstein of the Johns Hopkins Oncology Center in Baltimore had successfully demonstrated that different types of cancer presented a unique set of mutations in p53, probably due to different agents, such as as the chemicals contained in tobacco smoke or the sun's UV rays.

Stratton – a young geneticist looking for mutations in muscle and other soft tissue cancers – was intrigued by the results.

"These are very important articles that suggest that, yes, the mutagens that cause cancer mark the genome," he recalls. "It had a big impact on me as an opportunity for genomics, but it had to stay in the locker for 15 years while waiting for the technology."

In 2009, he and his team produced the first complete sequences of the cancer genome. These were detailed maps showing all the genetic changes and mutations that occurred in two individual cancers – a melanoma of the skin and a lung tumor.

These types of cancer choices were far from random: decades of epidemiology and laboratory studies have shown that exposure to UV light is probably the only cause of melanoma, while the link between tobacco and cancer of the lung goes back to the 1950s. Stratton and his team had a better chance of finding clear mutant fingerprints in the genome. But although they expected to see the same types of mutations in the genomes that Harris and Vogelstein had already taken in their studies on a single gene, they were not prepared for it. the sheer scale of genomic vandalism that they have discovered.

"Melanoma had something like 25,000 mutations, which was more than the world had ever seen in a genome," says Stratton. "We could really see the signings of the exhibits that had occurred at incredibly fine resolution, and we could see all kinds of features and nuances that we had never noticed before."

In the same way that a human fingerprint is a mixture of different types of peaks, mutant fingerprints consist of characteristic patterns of changes in DNA.

Carcinogenic chemicals cause mutations by physically binding to specific bases and affecting their shape. These alterations create a molecular key in the work, delaying fundamental processes such as copying DNA or reading genes. They must be repaired to keep the cell healthy and function properly. For example, benzo (a) pyrene (one of the main carcinogens of tobacco smoke) tends to attach to G bases, as does aflatoxin, a carcinogenic chemical produced by certain molds. But each of these types of damage is repaired in a specific way, leaving a characteristic change in the DNA sequence.

On the other hand, UV light causes mutations by blocking neighboring Cs. When the DNA copy machine encounters these merged pairs, it interprets the unusual form as a pair of Ts, causing a permanent change in the DNA sequence in that position.

"In order to analyze and distinguish these causes, we need to have a way of classifying mutation patterns, much like identifying a specific set of fingerprints according to particular patterns of loops and turns", explains Stratton.

Initially, Stratton and his team focused on six basic mutation signatures: C to A, C to G, C to T, T to A, T to C, and T to G. But there are several different mutation processes. a C to a T, which makes it difficult to say what may have been the underlying cause. The researchers then realized that some mutations tend to appear in the context of certain DNA sequences, due to specific chemical interactions or biological machinery at work.

There are also other distinctive changes in the genomes of cancer cells – including deletions or insertions of small sections of DNA, characteristic changes in consecutive base pairs and larger alterations and rearrangements – which can help with particular mutation process.

The genomes of melanoma and lung cancer were powerful evidence that fingerprints of specific culprits could be observed in cancers with a major cause. However, these tumors still contained many mutations that could not be explained by UV or tobacco, while what caused them? And what about cancers with no single cause so obvious? With thousands of mutations in a typical tumor, detective work becomes much more complicated for cancers with complex, multiple or even completely unknown origins.

As an analogy, imagine that you are a forensic scientist who strips fingerprints on a murder scene. You could be lucky and find a set of perfect prints on a window or door handle that matches a known killer in your database. But you're much more likely to discover a mixture of fingerprints from a range of people – from the victim and potential suspects to innocent parties and police investigators – all on all kinds of surfaces. .

Fortunately, Ludmil Alexandrov (now an assistant professor at the University of California at San Diego), a doctoral student at Stratton, has found a way to solve the problem. He realized that individual mutation signatures in a tumor can be distinguished from each other using a mathematical method called blind source separation, previously used to separate data from multiple sources, for example by separating the vocal and instrumental tracks of a single audio file.

In 2013, the Sanger team used a version of this technique to extract 20 distinct mutation fingerprints from nearly 5 million mutations in more than 7,000 tumors, covering 30 of the most common cancers. Some fingerprints were found in each tumor, while others were specific to a handful of cancer types. All cancers had at least two different fingerprints, while some had at least six.

Some of them come from things we already know to significantly increase the risk of cancer – the usual suspects such as tobacco or PAHs (polycyclic aromatic hydrocarbons, released during the burning of certain materials). Some previously suspected carcinogens have also been confirmed as hazards, such as aristolochic acid, a chemical produced by plants commonly used in herbal supplements in Taiwan and elsewhere. Other fingerprints are signs of internal work, resulting in fundamental processes of life inside our cells, including DNA copying and repair.

But the causes of about half of these fingerprints remain a mystery, left in the genomes of cancer cells by the culprits still on the run.

Find factors on the ground

The endoscopy room of the teaching and reference hospital Moi of Eldoret, in western Kenya, is a very busy place. Esophageal cancer is one of the most prevalent tumors in the region and every day, a seemingly endless stream of patients comes in search of relief from the salient blockages of their chutes. Most have not eaten well in weeks, making them incredibly thin and fragile. Some have come hundreds of kilometers from outlying rural areas, spending valuable amounts on expensive rented transport. All are desperate for help and most will die in the next year.

I look at the small pink blood cells of cancerous tissues that are carefully brought into plastic jars and sent to a freezer in a building on the other side of the hospital, waiting to be shipped to IARC. The on-site team will purify the valuable DNA of each sample and then send it to the Sanger Institute for it to be sequenced and analyzed to detect any mutational signature likely to be detected. explain what causes all these cancers.

I am here in Eldoret to meet Diana Menya, a Kenyan epidemiologist who has been working at the ego for many years. She is generous and in a good mood, with contagious energy. We feel like old friends after spending two days on a minibus – including an impromptu trip to see teenagers throwing themselves into a crocodile-infested pool – and she's full of laughter and stories about the area and its people . It is this curiosity and passion for his region that first made him aware of the exceptionally high rates of cancer in the area.

"A few years ago, I noticed that there were a number of patients with difficulty swallowing, and when the diagnosis was finally made, it was a matter of fact. a squamous cell esophageal cancer, "says Menya. "We were seeing more and more patients arriving at the hospital and I was wondering: what is it? What's going on here? Something has to happen. be done."

The Menya study has already shown that tobacco and alcohol are two of the factors likely to be at the origin of the overabundance of esophageal cancers in the United States. Western Kenya, which is not surprising, as previous epidemiological studies have linked them to. But although these two culprits may be at the root of many cases, they certainly can not explain them all.

To go out in the rural community outside Iten is to walk in a world of carcinogens. Farmland can be lush and fertile, but it is also flooded with pesticides and fertilizers that can seep into the water (usually unfiltered). There are cabbage fields that grow in a dish known locally as sukuma wiki, which is particularly rich in nitrates, which can then be turned into carcinogenic nitrosamines in the body.

We visit Emily in her kitchen – a single unventilated room covered with a thick layer of soot that falls back in places like stalactites. The smoke from the open fire is very strong and it is impossible to stay there for more than a few seconds without rushing. And where there is smoke, there are PAHs released from the burning of fuels such as wood, corn cobs and cow dung. Women, girls and children are particularly vulnerable because they spend a lot of time in the kitchen, often sleeping at night to stay warm and safe.

Corn is a common source of food and fuel in this region, and ears and almonds are often treated with a fungicide to prevent the growth of toxic pink molds (which can themselves cause cancer). Burning these chemicals with the spikes can release other carcinogens in the unventilated atmosphere.

Then there are personal habits. It is common in East Africa to drink extremely hot tea, sipped at temperatures up to 70 ° C (something I found out the hard way). Very hot drinks have already been associated with esophageal cancer in Iran and in parts of South America.

Poor dental hygiene could be another factor. A study conducted in China showed that the less teeth a person has, the higher their risk of esophageal cancer, perhaps because of the leaching of toxic bacterial chemicals in the saliva of infected gums.

While it's easy to suspect all these things (and more) of being at the origin of high rates of esophageal cancer in Kenya, we still do not have enough data to link most of these factors to footprints left in the cancer genome. This means that we do not yet know which of them are the most dangerous or how they can act together to cause the disease. In the future, we should be able to associate more carcinogens with their fingerprints by combining the mutography approach with in-depth epidemiological studies such as ESCCAPE. But this is not the only way.

Rather than studying DNA-extracted tumors to look for mutation signatures, David Phillips, professor of environmental carcinogenesis at King's College London, tackles the problem in the opposite direction. As part of the Mutographs project, he and his team are treating lab-grown cells with DNA-damaging agents and sequencing their DNA to see what mutational signatures have been left behind.

"By systematically examining human tumors and comparing them with mutation signatures in experimental systems caused by substances that we think or know to be carcinogenic to humans, we can compare them and say" Aha! the chemical is involved, "explains Phillips. "We work independently through things we suspect or know about causing human cancers and see what signatures we can draw from them."

Until now, Phillips and his team have tested 80 suspected causes of DNA damage, about half of which produce distinct fingerprints in the genome. Some are known human carcinogens, such as UV rays and aristolochic acid, which produce models of damage that might be expected given their properties. Whenever these appear in Mutographs tumor specimens, it is a safe bet that the agent involved is involved somewhere.

But he found other chemicals that leave mutation signatures in cells grown in the laboratory that have not yet been detected in human tumors. Maybe these molecules are truly carcinogenic, but it's rare that people are sufficiently exposed to be able to appear in their cancer cells, or maybe they're really innocent and can be excluded from Stratton's investigations.

It's a bit like catching someone who's rolling in a car, taking his fingerprints and putting them in a database: maybe they'll never commit a more serious crime, but if they do, the police will be much more likely to catch them. .

Save the future

At this moment, we are still in the first pages of this police story. A year after the start of the mutography project, a full team has been formed, the tools are in place and researchers are beginning to collect and analyze the fingerprints of cancers around the world. The scale of the project is staggering.

"As we refined the sophistication of the approach and the classification of mutations, algorithms and sequencing, it became apparent that it was a big challenge requiring investment and a coordinated organization, "says Stratton. "We have to collect five to ten thousand tumor samples and normal blood, we need to control the quality of DNA sequencing and manage the data and statistics – it's a combination of epidemiology to large scale and large-scale genomics that has not been married together that way before. "

In June 2018, researchers from the Mutographs team met at the Sanger Institute to share preliminary data on the first cancer genomes to get them through the project pipeline. Curiously, the first oesophageal tumors in Kenya do not seem to have PAH signatures, potentially highlighting smoky rural kitchens like Emily's, though it's important to point out that only a small fraction of cases has been analyzed so far.

Although it is too early to determine with certainty the suspects, in a turn of conspiracy worthy of Agatha Christie's "Murder on the Orient Express" – the cancer genomes clearly show that we are not in danger. We are not dealing with individual villains but a bunch of miscreants, each of them administering a potentially fatal blow to the genome. Everyone causes chaos in his own way, but they can combine to cause a catastrophe.

The flip side is that it is almost impossible to identify a specific cause of a given tumor or to say exactly what the cause is. A cell can be riddled with mutations, accumulated from all sorts of processes in its lifetime, but if none of them hit the vital genes or control switches responsible for growth or death, it will remain healthy. And because every cancer genome is traversed by thousands of mutations, it's impossible to say which culprit carried the coup de grace. But we should be able to draw a much more accurate picture of the contributions of different factors – be they biological or environmental – to each individual's illness.

This research comes too late to help Emily's parents, lying in their graves on the hillside. But Diana Menya hopes her work will save lives in the future.

"I think it's a combination of environmental factors and behavioral factors – something to do with what people do, what they eat, how they live and their habits, such as smoking. and alcohol, "she says, reminding women like Emily and her smoky kitchen. She also hopes that the project will distinguish the roles of nature and those of the esophagus cancer epidemic in Kenya. "We have anecdotal information in the families of our study site, but is it genetics or is it a shared environment? I hope the study on mutographs will answer that question."

This is true prevention research – discovering what increases the risk of cancer at a fundamental level, then using knowledge to sustainably bring about life saving changes in public health. But it takes time and requires political will. Menya has already seen this approach pay off by leading a campaign to support the extermination of Guinea worm all over the country.

"Pour prévenir une maladie, nous devons prendre des mesures. Nous avons éradiqué de nombreuses maladies, en particulier les maladies infectieuses. Ne pouvons-nous pas éliminer les maladies non transmissibles comme le cancer?" Menya dit. "Cela va sembler très ambitieux, mais pour moi, le meilleur résultat sera un Kenya sans cancer en empêchant les cancers de se produire en premier lieu – avant de les attraper".