Author:

Rachel Brazil

Abstract

Human papilloma virus is the causative factor of >90% of cervical cancer and few other tumour types. In this narrative review, the role of European research in identifying HPV function in cancer development, screening and prevention is illustrated through the voices of 3 key players, that actively contributed to the launch of the 2018 WHO campaign to eliminate cervical cancer.

One of the most impactful discoveries in cancer research over the last half century has been the link between the Human Papilloma Virus (HPV) and cervical cancer. The resulting vaccine, which has been available since 2006 is now routinely given to girls in at least 125 countries and is on the World Health Organization's List of Essential Medicines. Where it has been introduced, cases and deaths from cervical cancer have dropped - a 2019 study of 66 million young people worldwide found that the vaccine reduced pre-cancerous cervical lesions in young women by more than 50 percent. Much of the work to establish the causative link between HPV and cervical cancer was carried out in Europe. In 2008, virologist Harald zur Hausen was awarded the Nobel prize in Physiology and Medicine for his pioneering discoveries.

Zur Hausen passed away in May 2023, but the previous accounts of his work explain how his discovery of this association were met with considerable pushback for some time. The idea that cancer could be caused by a virus was not new. As far back as 1911 the retro-virus Rous sarcoma (RSV) was linked to cancer in animals. But by 1961 when zur Hausen started to examine if a vaccinia (cowpox) virus could produce chromosomal breaks in mouse cells, there was little interest in viral links to cancer from others.

Early in his career, zur Hausen post-doced in Philadelphia in the lab of Werner and Gertrude Henle who were studying the Epstein-Barr virus, a sub-type of the herpes simplex virus (sub-type 4) which had been observed in cultured Burkitt’s lymphoma cells. But zur Hausen did not see evidence for the persistence of such infections leading to viral transmission as the Henles supposed. In an interview with Cancer World in 2005, zur Hausen explained that he had started to think that cancer was linked to the spontaneous activation of the virus in only a few cells, based on similar observations in lysogenic bacteria. In his own lab at the Institute of Virology in Würzburg, he was able to show that genetic material from viruses like Herpes Simplex (HSV) can persist in human cells and only much later cause tumour growth.

For those who were studying viral links with cervical cancer in the 1970s, the focus of research was the HSV sub type 2. By 1972, zur Hausen, then at the University of Erlangen-Nuremberg in Bavaria, was not convinced of this link but started to examine a range of candidates, including the human papilloma virus (HPV), responsible for skin warts. ‘His hypothesis was that, unlike what 95% of the community was thinking, it wasn’t a herpes virus… he was challenging this view and pushed forward the hypothesis that human papilloma viruses are responsible for this disease,’ says Lutz Gissmann now retired as head of the division Genome Modifications and Carcinogenesis at the German Cancer Research Center (DKFZ) in Heidelberg.

Around this time, Lutz Gissmann began working in zur Hausen’s lab to differentiate the many HPV sub-type (now over 100) and discover which, if any, were associated with cervical cancer. In 1977 Gissmann moved with zur Hausen to the Institute of Virology at Freiburg and here he was able to extract viral genetic material from warts caused by HPV and compare these to genetic material found in cervical cancer biopsies. ‘I went to genital warts, and was able to find different HPV types, type 6 and 11, but they were not present in cervical cancer’ says Gissmann, although they did find distantly related sequences in cervical cancer biopsies.

Together with Gissmann’s PhD student, Matthias Durst, now at the Friedrich Schiller University Jena, Germany, they started using new methods to amplify the genetic material present and were able to identify first HPV subtype 16 in 1983 and then in 1984 with Michael Boshart, now at LMU Munich, HPV 18 as the two high risk papilloma viruses; HPV 16 being present in about half of cervical cancer biopsies and HPV 18 in a further 17%–20%. ‘It was clear that practically all cervical cancers were associated with one of these so-called high oncogenic papilloma viruses, says Gissmann.

Gissmann says their lab shared molecular clones of these newly identified HPVs to others studying the papilloma viruses including pathologist Peter Howely at Harvard Medical School who confirmed the presence and expression of human papillomavirus 16 and 18 sequences in human cervical carcinoma cell lines.

But by this time PCR was making genetic research much easier and there were multiple reports of HPV 16 in many tissues leading to a period of confusion and scepticism. ‘This did not hit me really,’ says Gissmann, ‘but it did affect zur Hausen,’ who in 1983 had become director of the German Cancer Research Centre at Heidelberg. Gissmann does recall a herpes simplex conference where the majority of researchers were still linking cervical cancer to the HSV type 2 virus. ‘[zur Hausen] stood up in this HSV conference and said no, what you're saying is all rubbish. I have here the better evidence.’ But this was not well-received.

This changed in the 1990, when epidemiological studies started to confirm that papilloma virus was the causative agent. One of those involved in this work was Francesc Xavier Bosch, who was an epidemiologist in the International Agency for Research on Cancer (IARC) in Lyon from 1982-1993 and served as Director of the Cancer Epidemiology Research Program of the Catalan Institute of Oncology in Barcelona, Spain, from 1994-2015.

From the late 1980s Bosch and colleagues in Spain and Colombia set up a case control study to investigate the role of men in transmitting the risk of cervical cancer to women. They came across zur Hausen and Gissmann’s work suggesting the link to HPV, so they added protocols to collect biological specimens to look for it. ‘It was a significant advance over studies based on questionnaires / serology.’ says Bosch. The advent of DNA sequencing, using early versions of PCR, allowed them to move from simple risk analysis to studying viral DNA in a person’s cells on a population level - a step forward into ‘molecular epidemiology.’

His team analysed the entire collection of specimens from two case control studies in Colombia and Spain, and in 1992 published results showing that the link between HPV and cancer was uniquely strong and provided very consistent evidence of causality. ‘Within a few years all previous associations were re-evaluated under the light of the dominant factor, HPV’ says Bosch. In an interview by the International Papilloma Virus Society in 2019, he previously recalled the moment of confirmation. Late on a Friday night he received a call from a colleague in Baltimore, Keerti Shah, who was in charge of the lab analysis of the specimens. ‘I jotted down some key figures on the back of an envelope, and after a quick calculation we realized this is it!… we were awestruck by the magnitude of the proof we held in our hands.’

This was followed by a campaign to spread the news and Bosch remembers clearly another ‘wow moment’ during a workshop at the University of Brussels. ‘After three long days of consultation in a dank, gloomy university facility, the late Dr. Shah turned to the audience and softly said, “Causality has been proven beyond a reasonable doubt”. That was a profound moment I shall always remember,’ said Bosch. After their findings were confirmed across a set of studies that examined specimens for over 20,000 cancer cases worldwide, they were ready to claim that not only was there a causal link, but that HPV was “the necessary cause”. They concluded that all cervical cancer cases are caused by HPV and therefore the prevention of HPV should control cervical cancer.

At this point Bosch says “the field was excited, and the annual meetings of the HPV group (latter became the HPV society) were an example of international collaboration, exchange of ideas and friendship. It helped that the key results were consistent in all parts of the world and there was no significant finding that challenged the central hypothesis on causality”. In 1995 the IARC produced a monograph involving 100 experts, reviewing the literature evidence and formally establishing HPV 16 and 18 as “group A” human carcinogen.

Since the link was established there has been further work to understand the mechanism involved. A small number of individuals who are infected by the oncogenic HPV strains remain chronically infected, which over time introduces cellular changes. Several viral proteins (E6 and E7) have been implicated as well as changes to two critical genes p53 and Retinoblastoma, which disrupt the control of cell growth. “This process takes time, often decades, from infection, which is very common in the young/adolescent age groups and the cancer incidence typically occurs in the 40`s and 50’s age groups” explains Bosch.

He says the discovery of the causal link suggest there should be a move towards testing for the virus rather than the conventional pap smear approach which examines cells for cancerous or pre-cancerous growth, but Bosch says even by 2001 there was still some push-back by the FDA to modifying what had been the standard screening practice over the last 50 years. “HPV screening has a significantly greater sensitivity than the cytology tests (some 20-30% increased detection of prevalent lesions) allowing for more clear reassurance of normalcy and longer intervals between screening events in HPV negative women” explains Bosch. But the application for a licence for the first clinical HPV test was initially turned down. Bosch was then commissioned to set out the evidence for causality again and once this was added to the dossier, approval was given and it was clear that there was now full consensus globally.

While there are still differences in how screening programmes are carried out, the most important next step was the development of a vaccine which in 2006 became available in the US, marketed by Merck & Co. under the trade name Gardasil. The vaccine was developed by Ian Frazer and Jian Zhou at the University of Queensland, Australia and received EMA approval in 2007. It is estimated to have prevented 17.4 deaths per 1,000 adolescents vaccinated, and prevents up to 90% of all cervical cancer cases. Gissmann says there was also work on a prophylactic vaccine for those already infected with HPV but it never gained traction. “There was lacking interest from the industry because they said we have a new vaccine, so why should we bother to invest in this” he says. So whether such a therapeutic is possible is still an open question.

Efforts now are focused on extending the current vaccination programme globally. “In [Western] societies, the screening works relatively efficiently, so the incidence of cervical cancer are relatively low, maybe 80/100,000 per year, whereas in developing world countries its five to 10 times higher” says Gissmann. Cervical cancer is the most common cause of female cancer death in nearly half of sub-Saharan African countries and global vaccination coverage rates for girls remain low (12% in 2021), with low rates also found in South-East Asia.

The WHO is now trying to completely eradicate cervical cancer within the century. “Vaccines are likely to be the pivotal intervention and screening an essential component to accelerate the cancer mortality reduction,” says Bosch, but he also points out, “cancer treatment is strongly related to the health care resources of the country.” Part of the task will also be education, of both the public and other health professionals and at the Catalan Institute of Oncology in Barcelona Bosch and colleagues have built an e-oncologia educational program, including courses on HPV and cancer prevention in seven languages.

This huge advance in cancer prevention is strongly associated with European research labs. “Some of the critical studies (case-control and cohort studies as well as international prevalence surveys) were strongly supported by European sources and European research groups,” says Bosch. Zur Hausen had previously suggested that at the time he was shifting the direction of his research towards HPV, US interests and funding were shifting away from supporting research into viral causes of cancer. But Gissmann says much of the interest in Europe can be laid at the feet of zur Hausen himself and his persistence in the face of others scepticism.

The now clear evidence for the link between HPV and cervical cancer has led to further links between HPV and vulva, vagina, penile, anal and oropharyngeal cancers. Gissmann says these associations are more difficult to prove because cervical cancers alone develops from well-defined precursor lesions that can be sampled for HPV. ‘These precursors don't exist in oropharyngeal Cancer, [but] everyone's expectation that in 20 years or so, in a vaccinated population, oral-pharyngeal cancer incidence will also drop, but you could never make this clear in a clinical study.’ Gissmann adds in 20 - 40 years if we suddenly see a decline in other cancers in vaccinated populations, we may even discover other cancers that are linked to HPV.

But there are still many unanswered questions regarding the mechanisms that allow HPV infection to lead to cancer many years after the initial infection. Zur Hausen pointed out that while men are also infected, rate of penile cancer are very low compared to cervical cancer, He wondered if oestrogen had a role in the ‘immortalisation’ of infected cancer-causing cells. Until his death in 2023, he continued to look at the role of infectious agents in cancer, focusing on whether plasmid like-DNA molecules known as "’Bovine Meat and Milk Factors’" (BMMFs), that infect beef and cow's milk are linked to a higher risk of developing colon and breast cancer. They contain sequences similar to the genes found in viruses, that allow them to replicate and zur Hausen hypothesised that like HPV they may be responsible for triggering cancer decades after infection.

Gissmann says the work he did with zur Hausen on the link between HPV and cervical cancer was some of the most important of his career. ‘When I think back on my scientific life, this was very rewarding because I could follow the whole history, I was involved in identifying this link and then seeing how it was developed and the current clinical effects, not many researchers get that.’