Warts and all

This essay was written by John Doorbar and was first published in the 1999 Mill Hill Essays. An updated version was published in the Mill Hill Essays anthology. 

A recent exhibition at the Museum of London displayed Oliver Cromwells Death mask. Oliver Cromwell suffered from warts and a number of these are prominently visible on the mask. They are also apparent in paintings of Cromwell and he is reported to have asked Peter Lely to paint him ‘warts and all’ in his portrait now displayed in the City Museum and Art Gallery in Birmingham. Cromwell tolerated his warts, possibly because the treatments available in the middle of the seventeenth century were often quite rudimentary. Application of plant extracts, paring with a sharp penknife and the use of corrosives such as brimstone are amongst the ‘cures’ described by Daniel Turner in his medical text De Morbis Cutaneis, which was published in 1712. Numerous folk cures were also available. In the sixteenth century, Sir Francis Bacon claims to have cured his warts by rubbing them with pork fat that was subsequently hung in the sun – as the fat melted the warts disappeared. Potatoes, green walnuts, broom straws or the intestines of a black chicken can be used instead of pork fat, but apparently these must be buried and left to rot after the wart has been rubbed.

Warts were also known to the Romans. The roman word for wart is verruca which means little hill or eminence. In 25 AD, Celsus in his book De Medicina suggested burning warts away with the ash of wine-lees, while Galen, during a visit to Rome observed a dextrous fellow who went about biting or sucking off verrucas from the feet of sufferers. The Romans suspected that genital warts were sexually transmitted but paid little attention to their cause. Daniel Turner in 1712 suggested that warts might be ‘congealed nutritious juices’ that had seeped from damaged nerve filaments in the skin. It was not until the end of the nineteenth century that the infectious nature of warts was recognised by the London physician Joseph Payne, who reported developing warts on his thumb after scraping those of a young patient. The suggestion that warts were caused by a virus came soon afterwards, when it was shown that a filtrate of common wart extracts could produce warts in the skin of volunteers. The concept was not generally accepted until 1950 however, when papillomavirus particles were actually visualised using the electron microscope.

These early images showed that the papillomavirus has a twenty-sided structure and is so small that four million would fit into the space occupied by the following full stop. It was another thirty years before the first sequences of papillomavirus genes were obtained and the great diversity amongst papillomavirus types revealed. Cromwells’ warts were probably caused by human papillomavirus type two, which is widespread in the general population where it usually causes warts in children. Around one in ten schoolchildren suffer from warts, and human papillomavirus type two is the most common cause. More than one hundred different human papillomavirus types are now known to exist, and each has a preference for a particular type of skin surface either inside or outside the body. Human papillomavirus type one likes to infect the soles of the feet and the palms of the hands and is the usual cause of verrucas. Types four and sixty-five also do quite well here but the damage they produce looks a little different. Some papillomaviruses have peculiar distributions which are not easily explained. Human papillomavirus type seven causes butchers warts which are found predominantly in poultry workers and meat handlers, while human papillomavirus type five seems to turn up only in individuals whose immune system is compromised, and can therefore be a major problem for transplant patients. Human papillomavirus type five and its close relatives, types eight and fourteen are not clearly associated with disease in the general population although presumably there is a reservoir of infection here. Yet other types specialise in the infection of mucus secreting surfaces such as the cervix. Genital warts caused by types six and eleven are the most common sexually transmitted disease seen in the United Kingdom and affect one in two hundred people between the ages of twenty and twenty-four.

Papillomaviruses are medically important partly because they cause genital warts but more specifically because they are associated with cervical cancer. Over ninety-five percent of cervical cancers have been shown to contain papillomavirus DNA – usually from just a small number of human papillomavirus types which are now regarded as ‘high risk’ viruses. The most prevalent of these is type sixteen which is found in over half of all cervical cancer cases, with three other types, eighteen, thirty-one and forty-five, accounting for most of the rest. The commonly-used Hela laboratory cell line was derived from a cervical tumour in 1952, and continues to grow in the laboratory because it contains nucleic acid from human papillomavirus type eighteen. Other laboratory cell lines contain nucleic acid from human papillomavirus type sixteen and were derived from similar sources in the nineteen seventies. Of course human papillomavirus types sixteen and eighteen don’t ‘want’ to cause cancer, and in most cases they don’t. From the point of view of the virus, the most satisfactory outcome of infection is that a wart forms and new infectious viruses are shed from the surface of the skin. Human papillomavirus type sixteen infection normally leads to the formation of flat warts which are similar in appearance to the normal tissue of the cervix, and this is where type sixteen viruses are assembled during a productive infection. The more efficiently new infectious virus particles are made then the more chance the virus has of infecting someone else. When an infection with human papillomavirus type sixteen progresses to cervical cancer, new virus particles can no longer be produced and the control exerted by the virus over the cell is lost. Progression to cancer benefits neither the virus nor the human host.

Why should a small group of papillomaviruses which infect the cervix be associated with a human cancer, while the ones which cause common warts and veruccas are harmless? Both type one and type sixteen papillomaviruses reproduce by diverting the normal replication machinery of the host cell, but type sixteen does this in cells of the cervix rather than those on the sole of the foot. As a consequence, its proteins have evolved to work best in that particular environment, and it is not too surprising that differences have resulted between the two virus types. Evolution would be expected to weed out the bad changes and fine tune the viruses to their respective cellular niches, and it is clear that human papillomavirus type one is well adapted to its target site. A single verruca can contain around one thousand billion virus particles and the abrasive flooring found in swimming pools helps their spread. The association of type sixteen with cervical cancers suggests that it is not perfectly adapted to its site of infection, and the reason for the problem can be attributed in part to just one of the virus proteins.

The first line of defence a cell has following infection by type one or type sixteen human papillomavirus is to commit suicide. Infected cells can sacrifice themselves for the good of the whole organism, thus stopping the virus from starting or maintaining an infection. Obviously this is bad news for the virus, and the papillomaviruses have evolved cunning counter strategies to avoid this cellular defence. For type sixteen virus, the critical event is the association of one particular virus protein called E6 with a protein called p53, produced by the infected cell. In normal skin cells the amount of the p53 protein increases following damage to the cellular DNA, as might occur during sunbathing, or when replication of the cellular DNA occurs at an inappropriate time such as following virus infection. This increase in the amount of p53 protein causes the cell to stop growing which allows the cell time to put things right, or induces it to commit suicide if the damage is too serious and cannot be repaired. However, when cells are producing high levels of the E6 protein as a result of infection with the types of papillomaviruses associated with cancers, the level of the p53 protein is kept down as a result of the E6 and p53 proteins sticking to each other. When this happens there is no arrest of cell growth or cell suicide and cancerous growth can result. Loss of p53 protein from infected cells presumably gives the virus an advantage during its normal life cycle, but it also means that cells infected with type sixteen virus lose a key defence mechanism. By contrast, the E6 proteins made by papillomavirus types which are not associated with human cancers, such as those which cause common warts, genital warts and verrucas, don’t stick to the p53 protein. In the small percentage of cervical cancers where there is no infection with a papillomavirus, the amount of p53 protein is elevated but the protein always contains mutations which prevent it from acting normally.

So how do these infections contribute to the development of cervical cancer? Compared to the number of women who are infected with papillomaviruses, those that go on to develop cervical cancer are quite small. The usual outcome of infection, whether by high or low-risk viruses is that the immune system attacks the damaged infected area causing the wart to disappear, leaving little trace that the virus was ever there. With common warts this takes on average around two years although in some people it can take much longer. This can occur with cervical infections too, and if it does then the risk of developing cervical cancer is largely eliminated. If a cervical infection persists then the DNA from the virus can become integrated into the DNA of the host cell. Nearly all cervical cancers are found to contain integrated viral DNA. During integration, large parts of the virus DNA are usually lost, but the gene which is responsible for making the E6 protein is always preserved, as is the part of the viral DNA necessary to make cells proliferate. A second clue as to how cervical cancer develops comes from examining the pattern of the disease. Human papillomavirus infection of the cervix peaks amongst sexually active women in their early twenties, and declines with increasing age. By contrast, invasive cervical cancer is most common in older women, reaching a peak around fifteen to twenty years later. The continued presence of the E6 protein and the resulting loss of p53 protein, results in an inability of the infected cell to repair chance mutations that can occur in the several decades that pass following the first infection. Anything that increases the chance of such genetic changes occurring, such as smoking which results in the presence of cancer-causing chemicals in all mucus secretions, including cervical secretions, will increase the likelihood of cancer developing.

In developing countries cervical cancer is the most frequent female malignancy, accounting for about a quarter of all cancers in women. The National Health Service cervical screening programme aims to detect cervical abnormalities before they become life-threatening. It was introduced as a national programme in 1988 and is credited with reducing the incidence of cervical cancer in the United Kingdom to around seven percent. Three and a half million women are invited for screening each year of whom a minority will receive an abnormal result. Screening relies on the fact that cervical cancer is usually preceded by a long time interval in which a series of defined cellular abnormalities can be detected using the standard Pap smear. The Pap smear derives its name not from the papillomaviruses which cause the disease, but from George Papanicolaou who developed the test during the late nineteen thirties while working at the Women’s Hospital in New York. It was first used for mass cervical screening in 1948 in the state of Massachusetts and the test has changed little since then. Cells are taken from the surface of the cervix, smeared across a glass slide and stained with a mixture of dyes designed to make detection of abnormalities easier. A specialist technician trained in the examination of cells then examines the slide for abnormalities, such as cells that have enlarged nuclei or irregular shapes. Although this may sound simple, a typical smear contains around three hundred thousand overlapping cells of which only a small number may be abnormal, and the identification of these is often made more difficult by the presence of blood, mucus and drying artefacts. The subjectivity of the test and the limited amount of time available to screen each slide compounds the problem. A trained technician can reliably detect only around three quarters of precancerous cells and the test is associated with a high rate of false negatives. The percentage of women with abnormal smears who are given the all clear ranges from ten to forty percent. It is a bit like searching for a needle in a haystack and it is not surprising that problems associated with cervical screening are so often in the news.

Quite a lot has been learned from cervical screening failures. The worst was in Kent in 1996, when ninety-one thousand smears had to be retested. Three hundred and thirty-three women were found to have high grade abnormalities, thirty needed hysterectomies to halt the cancer and eight have since died. A year earlier, a single screener was found to have been responsible for three hundred and fifty-seven misread smears carried out over three years in Great Yarmouth. Nineteen of the smears were subsequently found to show serious abnormalities. Rigorous guidelines have since been introduced in an effort to ensure consistency between laboratories, and to reduce human error. These limit the maximum number of smears per screener per year to seventy-five thousand, and no more than eight smears per hour for no more than four hours at a time. Although this will undoubtedly help, it is clear that the methods used to predict cervical cancer need to be improved. Of the three and a half thousand or so women who develop cervical cancer each year in the United Kingdom, around thirteen hundred die because it was diagnosed too late. Cervical cancer is almost always curable if diagnosed early.

There are two obvious ways to improve things. Prediction can be improved or infection by the virus can be prevented. Several companies have developed image analysis systems which can identify abnormal cells automatically. When used in conjunction with traditional screening these can reduce false negative rates by up to half. Cervical screening currently costs one hundred and thirty-two million pounds each year in England and Wales, and cost effectiveness is a consideration when considering such options. Another way is to make the rare abnormal cells stand out better so that it is harder for the screener to miss them. Cell division in the upper layers of the cervix is an indication that something is wrong and such dividing cells can be specifically stained using antibodies. Cellular proteins such as MCM 2 and cdc6, are involved at the start of the cell division process, and their use in the identification of cervical abnormalities made national news last Autumn. The detection in cervical cells of DNA or proteins originating from the virus is another option. One US company actively markets a viral DNA detection system designed to distinguish between high and low-risk viruses in cervical smears. The idea is that high risk infections such as those caused by type sixteen virus are managed differently from infection with low risk viruses such as those caused by type six. The complication is that as many as a quarter of women have cervical papillomavirus infections at any one time, often without active virus production, and the presence of virus DNA alone cannot be used to predict future progression to cancer. A possible solution is to combine the detection of virus proteins which would be lost during cancer progression with the appearance of the cellular proteins which become activated. Only one protein, called E4, which is characteristic of virus infection is easily detected in the surface layers of papillomavirus-infected cervix, and its presence indicates that things have not yet become serious. As the severity of disease increases, the E4 protein is no longer produced and staining for the MCM protein produced by the infected cell becomes more diagnostic of disease. Approaches like this should allow abnormal cells to be highlighted during the routine Pap screen and also confirm the presence of a high risk virus infection at an early stage.

Over the next few years, advances in screening are likely to vie with each other for superiority until, it is hoped, one approach comes out on top and is incorporated into routine use. In many countries of the world though, an expensive screening programmes is not an option, and cervical cancer will remain a major killer for many years to come. In these situations, the solution may be to circumvent the need for screening by eliminating the virus. This could be achieved by stopping the viral E6 protein from blocking the cellular suicide response which should cut in following viral infection. Preventing E6 from working should lead to the death of infected cells. A better solution however may be to vaccinate against papillomavirus infection. Papillomavirus particles contain two proteins which wrap around the virus DNA. Production of these proteins in yeast or insect cell cultures leads to the formation of particles which closely resemble authentic viruses, but without the DNA that is required for new virus production. Vaccination of dogs with these virus-like particles has recently been shown to protect them against infection with canine oral papillomavirus, and trials in humans are currently in progress. Although this approach looks promising, it depends on the generation of large amounts of antibodies at the mucosal surfaces that papillomaviruses infect and this may be difficult to achieve over long periods. Therapeutic vaccines, designed to eliminate existing infections are also the subject of much current research. Most aim to generate immunity to the viruses which cause genital warts as well as to those which cause cancers. Such approaches should ideally stimulate our immune system, particularly at the mucosal sites of infection. Although human trials have yet to be started, studies on domestic rabbits infected with cotton tail rabbit papillomavirus have suggested that such approaches can be effective in eliminating existing infections.

It is now clear that most cervical cancers are caused by an infectious agent and a fairly simple one at that. Human papillomaviruses only have nine genes and with these they manage to take over specific skin surfaces and propagate themselves. Worldwide they are responsible for around four hundred thousand cases of cervical cancer each year. Successful intervention would not only save lives but would fundamentally change womens health care, and trials to improve cervical screening and to test vaccination strategies are already underway. The next few years should show if our optimism is really justified. A lot has been learned since Daniel Turner proposed that warts were ‘congealed nutritional juices’. If Cromwell were alive today we would probably suggest that he had them surgically removed or that he used Compound W or Bazooka to cure his warts, the modern day equivalent of brimstone or the ash of wine-lees. Plant extracts such as those derived from willow bark may have helped as it contains salicylic acid, the active ingredient in Compound W, but the benefits of pork fat are uncertain. There is still no clever cure for papillomavirus infections, but the realisation that certain types of the virus are associated with human cancer has greatly accelerated the search.

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