Unravelling the genetic causes of cancer

In addition to providing a major weapon for tracking and controlling the spread of drug-resistant bacteria, DNA sequencing is also being harnessed as a tool for cancer research. In 2012 the International Agency for Research on Cancer estimated that there were about 8.2 million deaths from cancer across the world annually­, i.e. approximately 22,000 deaths a day. Cancer accounts for one in seven deaths worldwide. The global burden of cancer is expected to rise to 21.7 million new cases and 13 million deaths by 2030 (American Cancer Society, 2015).

Cancer is a group of more than 100 diseases caused by mutations in the DNA code. This affects the making and working of proteins responsible for the growth and division of cells, directs where cells live in the body and determines when they should die. A major difficulty in tackling cancer arises because it is not usually caused by a single mutation in a single gene, but rather by multiple mutations that accumulate in several key genes. In addition, cancer mutates genetically over time, and changes in response to therapy. This makes the disease highly complex and heterogeneous. The fact that every cancer is different poses major challenges in terms of both its diagnosis and treatment.

This shows the evolution of cancer in cells. Credit: Adapted from J D Hunt (n.d.) 'An introduction to cancer', LSU Health Sciences Center. Cancer only develops when several mutations accumulate in the DNA of an individual cell. Most of the mutations arise in the tissue where the cancer starts. In this diagram the light grey cells represent normal cells from one of the body's organs. These may develop mutations that cause them to grow slightly faster than other cells around them. The light blue cells represent cells that have undergone the first stage of mutation. Such cells are not yet tumour cells and will only become so if they undergo further mutation, which is a long process. Once formed the tumour cells can further mutate and become malignant. These appear at the site of the original organ and undergo yet more mutations before entering the bloodstream and spreading to other organs in the body where they form metastatic tumours.

Sequencing the cancer genome

As early as 1986 researchers suggested that sequencing the human genome might provide a better means to understanding cancer (Dulbecco, 1986). From the late 1990s an international consortium of scientists from more than 70 countries has been involved in efforts to catalogue and analyse normal, pre-cancerous and cancer genomes. One of the first projects, known as the Cancer Genome Project, was set up by the Wellcome Trust and the Institute of Cancer Research in the UK in 1999. In 2006, a similar project, known as The Cancer Genome Atlas (TCGA), was established by the National Cancer Institute and National Human Genome Research Institute in the USA.

The international research effort is dependent on whole genome sequencing (WGS). Its objective is to improve knowledge about the genetic mechanisms underlying cancer and to identify and classify different cancer types so as to improve diagnosis and treatment. Much of the work is concentrated on the cancer genome, i.e. on all of the genes in a cancer cell. The first cancer genome was sequenced in 2008. Four years later, almost 800 genomes had been sequenced from at least 25 different cancer types. By 2014 over 25,000 tumour samples had been sequenced from 50 different types of cancer (Brice, 2014). In 2015 the TCGA announced that it had discovered nearly 10 million cancer-related mutations.

Challenges

Despite this achievement, however, genomic research has been controversial, in part because of the high costs involved. The TCGA alone absorbed US$375 million in funds between 2006 and 2015, which some researchers initially felt would be better spent elsewhere. One of the largest expenses in the project was finding and paying for fresh tissue samples, because originally the sequencing technology could only work on rapidly frozen fresh tissue. This was a problem because most clinical biopsies examined by pathologists were fixed in paraffin and stained, which rendered them unsuitable for sequencing (Ledford, 2015).

Sequencing the cancer genome has not only been questioned because of the large sums of money involved, but also because many have been sceptical about how far its results can be applied in the clinic. Only a small number of the mutations identified to-date have been found to induce cancer. Known as 'driver' mutations, most appear to be genetic oddities which have little in common. Even where driver mutations have been discovered, scientists have struggled to translate the findings into clinical applications. This has been particularly noticeable in the context of drug development. Cancers quickly activate different genes when drugs target their driver mutations (Ledford, 2015).

Inherited breast and ovarian cancer

While genomic sequencing as a whole is still in its infancy and its findings are still emerging, some progress has been made in the understanding of inherited breast and ovarian cancer. In fact, scientists had begun to understand the genetic mechanism involved in such cancers well before the launch of the Human Genome Project, and DNA sequencing.

The notion that breast cancer could be a hereditary disease was first put forward in the mid-nineteenth century by Pierre-Paul Broca, a French physician-scientist, based on his observation of ten cases of breast cancer in four generations of his wife's family. Decades later, in the 1920s, Janet Elizabeth Jane-Claypon, an English physician and one of the founders of epidemiology, showed that daughters of women who had died of breast cancer had significantly higher rates of mortality from the disease than those whose mothers had died of other causes. By the 1970s a number of epidemiological studies had established that the risk of breast cancer was much greater among the daughters and sisters of women who had died young from the disease (King, 2014).

Despite this evidence, not everyone was convinced that breast cancer could be an inherited disease in the 1970s. Indeed, many believed it was caused by a virus. This thinking began to change as a result of the work of Mary-Claire King, who was hired by Nicholas Petrakis at the University of California, San Francisco, in 1974, to work alongside his group of cancer epidemiologists to investigate why it was that some families were so severely affected by breast caner. While the evidence showed that breast cancer frequently appeared in multiple generations in some families, it was unclear whether this was due to their mutual exposure to some unidentified environmental factor, because of some inherited trait, or a combination of the two (King, 2005).

At the time King knew very little about breast cancer, but she soon became engrossed by the question of what caused the disease. Having worked on genetic issues relating to human evolution for her doctoral research, she found it natural to look at the evolutionary process of cancer. She decided to tackle this both from the familial perspective, looking at patterns of susceptibility, and from the point of view of the tumour, following its mutation, selection and migration process (Gitschier, 2013).

Mary-Claire King looking at an autoradiograph produced with the aid of Sanger's manual DNA sequencing technique. The photo was taken by Mary Levin in 1996. Credit: King and Washington University. King had first become aware of the devastation of cancer when, at the age of 15, she watched her best friend die of a kidney tumour. Her first degree was in mathematics, awarded to her when she was just 19 years old. Following this, she completed a doctorate in genetics in 1973 under the supervision of Allan Wilson at the University of California, Berkeley. She then moved to Santiago, Chile, to teach on a University of California exchange programme at the Universidad de Chile, but the programme was abruptly terminated after the country's socialist president, Salvador Allende, was overthrown by Chile's armed forces and national police in September 1973. This resulted in her return to the USA where she took up a temporary research position at the University of California in San Francisco before being appointed professor of genetics and epidemiology at the University of California, Berkeley in 1976. She remained there until 1995 when she moved to the University of Washington (McHale, 1996; King, 2014).

Initially, King set out to construct a genetic map based on evidence collected from women under the age of 55 diagnosed with primary breast cancer over a two year period between 1980 and 1982 in the San Francisco Bay Area and the metropolitan Detroit region of the National Cancer Institute's Surveillance, Epidemiology and End Results Program. The records contained no information about the family history of the women recruited to the programme. Such details were gathered by interviewing each case within 6 months of diagnosis to establish if any of their immediate female or male relatives had a history of breast cancer (Newman, Austin, Lee, King, 1988).

By 1988, King and her colleagues had analysed the genetics and mathematically modelled data gathered from 1579 nuclear families. Results from this study indicated that while 4 per cent of the families in the sample had inherited a susceptibility to the disease, the rest were afflicted by the disease by chance. Those who inherited the mutated gene were shown to have an 82 per cent life-time risk of developing breast cancer compared with 8 per cent of the general population (Newman, Austin, Lee, King, 1988).

The hunt for breast cancer genes

While the genetic mapping undertaken by King and her team indicated there was a strong possibility that breast cancer had a genetic link, this had yet to be proved. The best way to do this was to demonstrate the existence of such a gene. In 1988 a number of researchers in North America and Europe began competing against each other to find a gene dubbed 'Breast Cancer 1' (BRCA1) in the human genome. DNA sequencing and different genetic markers were crucial to this endeavour. The competitors included King's group as well as scientists under Bruce Ponder at the Cancer Research Campaign's Human Cancer Genetics Research centre based in Cambridge, UK, and an American group led by Mark Sckolnick who was linked to the University of Utah and Myriad Genetics Inc, a Utah-based biotechnology company.

In December 1990 King's team announced that the most likely place to find the gene was in a small region in the middle of human chromosome 17. They reached this conclusion on the basis of a detailed genetic analysis of 23 extended families, a total of 329 relatives, which had 146 cases of breast cancer. Each relative was expected to provide a blood sample, which was subjected to DNA sequencing. Findings from the study marked a critical turning point in the hunt for the gene. Nonetheless, the region identified was thought to contain hundreds of genes, so finding the right gene remained a major undertaking (Hall, Ming, Newman, Morrow, 1990; Arney, 2012).

By late 1992 another group of researchers based in England, led by the epidemiologist Doug Easton and funded by the Cancer Research Campaign, had completed a study analysing all the genetic linkage data collected from 214 breast cancer families from around the world; this sample included 57 individuals with both breast and ovarian cancer. This located the disease gene more precisely on human chromosome 17 and showed that a faulty gene in this region accounted for most of the cases of breast and ovarian cancer in the families. It also indicated that there could be two, not just one, gene involved. The evidence provided by Easton's group greatly narrowed down the scope of the search (Easton, Bishop, Ford, Crockford, et al, 1993).

In August 1994 Bruce Ponder's team in Cambridge published a genetic map they had constructed of the region of chromosome 17, indicating 20 possible genes linked to breast cancer. Following this, research teams across the world began sequencing DNA fragments prepared by Ponder's team from the region of the chromosome in an effort to find genes with the same mutations as those found in women with hereditary cancer. Within two months Skolnick's group in Utah announced that they had identified and cloned a gene (BRCA1) which was found in a high proportion of the families affected by hereditary breast and ovarian cancer (Miki, Swensen, Shattuck-Eidens, Futreal, et al, 1994; Arney, 2012).

While the discovery of BRCA1 by Skolnick’s group marked a major breakthrough, the research findings indicated that the gene was not implicated in all the cases of inherited breast and ovarian cancers. This suggested the involvement of another gene. In the same month an international team led by Mike Stratton at the Institute of Cancer Research, UK, with funding from the Cancer Research Campaign and the Medical Research Council, announced that they had located a second gene on a small region on chromosome 13 that predisposed individuals to breast cancer. This conclusion was based on the construction of gene linkage maps and the analysis of DNA taken from 15 families that had multiple cases of early-onset breast cancer not related to the BRCA1 gene (Wooster, Neuhausen, Manigon, Quirk, et al, 1994).

News from the Stratton team set off another international search to find the next gene linked to breast cancer. The main competitors were Stratton's group in collaboration with researchers at the Wellcome Trust Sanger Institute and Myriad Genetics. By the end of 1995 this team had identified the gene (BRCA2). They tracked this down after sequencing DNA samples taken from a number of Icelandic families who were affected by multiple cases of breast cancer. Access to a draft of the DNA sequence from the end of chromosome 13, provided by researchers in the Human Genome Project finally helped them identify the gene (Wooster, Bignell, Lancaster, Swift, et al, 1995; Arney, 2012a).

Subsequent to their discovery, both BRCA1 and 2 have been found to be pivotal to the production of proteins that help repair damaged DNA and sustain the stability of a cell's genetic content, an important mechanism in suppressing tumours. Women who inherit a mutant version of such genes run the risk of developing abnormal cells and cancer. Approximately 1 in every 1,000 people carry a fault in one of the genes, and about 2 in every 100 women with breast cancer have a fault in either of them. Women who have a faulty BRCA gene have an 80 per cent risk of developing breast cancer. This compares with a 12 per cent risk in the general female population. They also have a 55 per cent chance of getting ovarian cancer. Overall, mutations in both BRCA1 and BRCA2 account for approximately 20-25 per cent of hereditary breast cancers, about 5 to 10 per of all breast cancers, and 15 per cent of ovarian cancers (National Cancer Institute, n.d.; Arney, 2012a).

This shows two human breast cancer cells dividing. Credit: David Becker, Wellcome Images.

The prevention of breast cancer

The isolation and identification of the BRCA genes offered a means to determine an individual's risk of contracting cancer for the first time. Although a genetic test had been developed to identify individuals carrying mutations of the genes as early as 1996, many controversies surrounded its promotion. This was partly because many questions remained about the risk posed by the gene, and partly because it was unclear what, if anything, could be done to lessen the risk (Kolata, 1996).

Today genetic testing for BRCA1 and 2 is routine in the USA, being carried out mostly at the commercial laboratory of Myriad Genetics, while in Britain testing is recommended for women who have a strong family history of breast and ovarian cancer. King now argues that all American women aged 30 and over be offered the test. Not everyone agrees however Narod, 2009; Pollack, 2015).

Those who test positive face stark choices about what to do. Women in their twenties and thirties, for example, who have not yet been diagnosed with breast or ovarian cancer wrestle with decisions as to whether they should have their ovaries and breasts surgically removed. This would help considerably to reduce the risk of cancer, but could have potentially serious side effects, such as early menopause. In addition, if they test positive, they have to consider whether to share such information with relatives who may also carry the gene. All of this can take its toll emotionally (The Royal Marsden, n.d.).

The BRAF gene

Soon after identifying the BRCA2 gene, in 1998/99, Stratton and his colleagues Richard Wooster and Andy Futreal began to discuss the possibility of using the reference sequence data emerging from the Human Genome Project as a means to move beyond sequencing cancers caused by inherited mutations. Their idea was to look at cancer attributable to alterations to DNA that occur after conception. Known as somatic mutation, this process can occur in any cells of the body apart from germ cells (sperm and egg cells) which cannot be passed on to offspring. Their proposed project was highly ambitious. Not only was its scope large, the technology was still relatively undeveloped and it would require large-scale funding (Stratton, ­2013).

The team started work on what was to become known as the Cancer Genome Project in 2000 at the Sanger Institute in Cambridge, with funding from the Wellcome Trust. They began by sequencing some coding segments of DNA, known as exons, in cancer genomes. One of the obstacles facing them was how to get sufficient quantities of DNA to do the work because they could not secure enough normal tissue DNA samples from individuals whose somatic mutations had to be sequenced. This forced them to focus their attention on just 15 cancer cell lines. These provided an assortment of 20-30 cancers, including cancers of the breast and lung and melanomas. The work was carried out with the help of polymerase chain reaction (PCR), a technology that amplifies DNA (Stratton, ­2013).

The group was reliant on the conventional sequencing method developed by Sanger. This meant that they could not sequence large numbers of the genes in one go. Given this problem the group focused their attention on sequencing genes that coded for kinase enzymes, proteins that were known or thought to be involved in cell signalling pathways implicated in cancer development. They started the project by sequencing a large number of tumour samples which they then screened for any mutations linked to the kinase enzymes. Within weeks of starting the work they discovered that one kinase gene, known as BRAF, contained a single mutation that altered one amino acid in the majority of tumours. By 2002 they had demonstrated that BRAF mutations showed up in approximately 60 per cent of malignant melanomas, 15 per cent in colorectal cancers, 30 per cent in papillary thyroid cancers and a variety of other types of cancer (Davies, Bignell, Cox, Stephens, et al, 2002; Hesketh, 2012; Stratton, ­2013).

Mike Stratton (immediately in front) Richard Wooster (centre) and Andy Futreal (right), 2004. Credit: Sanger Institute. The three researchers are responsible for the foundation of the Cancer Genome Project and the discovery of the BRAF gene.

Malignant melanoma

The study was promising because it suggested that BRAF and its mutations could provide a drug target for malignant melanoma, a dangerous and highly prevalent form of skin cancer that is generally unresponsive to conventional chemo or radio therapy. In 2012, 234,000 people were estimated to have this cancer around the world, and it had caused 55,000 deaths (WHO, 2014).

Encouraged by the findings of the Cancer Genome Project, a number of pharmaceutical companies now launched work on the development of drugs to inhibit BRAF. The first one to gain approval from the US Food and Drug Administration (FDA) was vemurafenib (Zelboraf) in 2011. Another, dabrafenib (Tafinlar) was approved by the FDA in 2013. Both drugs provided a major step forward in the treatment of malignant melanoma, although as yet they do not provide a total cure, and many patients develop tumours that are resistant to the drugs. Attention is now being paid to alternative treatments targeting BRAF and its signalling pathway. This is being aided by the genetic analysis of patients who have proved resistant to treatment of vemurafenib and dabragenib (Fedorenko, Gibney, Sondak, Smalley, 2015).

Classification

While progress in the treatment of cancer still has a considerable way to go, sequencing has opened up a new approach to the disease. It has had a major impact in challenging the traditional classification of tumours, shifting classification to a system that is based on a tumour's genetic make-up rather than its location in the body. This is guiding new treatment strategies. Genomic analysis of tumours from patients with breast cancer, for example, has identified a number of driver mutations in genes not previously associated with breast cancer. Some of the genes found in breast cancer tumours have also been identified in leukaemia and cancers of the prostate, lung, skin, colon and rectum. The discovery of genetic mutations across different tumour types has also opened up the possibility of using drugs originally approved to treat other cancers. Such discoveries are also being used in the design of future drugs. WGS also makes it easier to know which patients are most likely to benefit from particular treatments (Hesketh, 2012; Chmielecki, Meyerson, 2014; Mardis, n.d.).

WGS is also proving valuable in tracking the evolution of cancer in tumours. Work in this area shows that when tumours grow they retain their original cluster of mutations and initially cause the cells to become cancerous; over time, they also develop new genetic mutations. This is important in terms of the timing of the administration of drugs, which are likely to be less effective if given in the late stage of cancer where more types of mutations need to be hit (Mardis, n.d.).

The dramatic drop in the cost of sequencing in recent years is fuelling expectations that doctors will soon be able to submit almost any suspected malignant specimens from patients for a full genomic evaluation as a matter of routine. A number of researchers are also investigating the possibility of developing a diagnostic test to sequence DNA originating from cancer cells that circulate in bodily fluids such as blood plasma. It is hoped that such tests will help in the classification of patients’ cancers, what their prognosis is, what treatment should be given, and the ability to monitor their responses to drugs (Murtaza, Dawson, Tsui, et al, 2013; Chmielecki, Meyerson, 2014).

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