In 1993, a pair of Brazilian researchers, Professor Luisa Lina Villa and Professor Eduardo Franco, began a prospective study, or cohort, aimed at understanding how infection by different types of human papillomavirus (HPV) leads to the development of cervical cancer.
The Ludwig-McGill Cohort Study followed around two thousand five hundred women volunteers for 5 years, with part of this group being followed for 10 years. Today, 31 years after the study’s inception, Professor Luisa Villa and Professor Eduardo Franco are transferring leadership of the project to Professors Laura Sichero and Helen Trottier, who have also been involved in the study at various stages of their careers.
C2PO spoke with Professors Luisa Villa and Laura Sichero about the study’s execution, its importance, and the leadership transition.
C2PO: How was the Ludwig-McGill Study conceived and developed?
Professor Luisa Villa: This cohort study began in Brazil, in São Paulo, when Professor Eduardo Franco and I were at the Ludwig Institute for Cancer Research. I was researching HPV, and Professor Eduardo was starting to study cancer epidemiology. We joined forces to design this study on HPV and cancer.
We asked ourselves what we knew about HPV at that time – in the early 1990s – and planned how to follow several Brazilian women over an extended period to understand the natural history of HPV and its relationship with cervical cancer.
There was very little knowledge on the subject when we started the cohort. This study was pioneering in many ways. In short, we sought to understand the following: What happens when a woman is infected with HPV? Does the infection persist or is it eliminated? If eliminated, how long does it take? Which women infected with HPV develop cervical cancer? What are the characteristics of these women?
By “natural history” I mean we made no interventions: the women lived their lives normally, and we collected samples, conducted tests, and tracked outcomes. A great deal of material was generated, processed, and stored. Participants were followed using many tools: questionnaires, HPV tests, cervical cytology, etc. When necessary, they were monitored and referred for treatment, especially in cases of high-grade cervical lesions.
The women returned for a visit every six months and were followed for 5 years. Some left the study over time, but the retention rate was very high: we started with about 2,500 women and finished the fifth year with around 1,900. Some were followed for up to 10 years.
Today, we no longer follow the participants, but we continue generating results from the work done 30 years ago.
C2PO: What were the major scientific findings, impacts, and contributions of the study over these 31 years?
Professor Laura Sichero: The contributions have been numerous. This study was developed to answer questions about the natural history of HPV infection and how the infection leads to the development of premalignant cervical lesions. It was crucial in evaluating the prevalence of HPV in the women studied, establishing how many of them had persistent infections, and determining which women developed cervical lesions.
One of the most important findings was that certain types of HPV, those considered high-risk, are associated with greater persistence of infection compared to low-risk HPV types. High-risk HPVs are also more associated with the development of precancerous lesions than low-risk HPVs.
Additionally, different risk factors for HPV infection and the development of cervical lesions were revealed – primarily sexual variables. For example, the prevalence of HPV is higher in younger women, those who had their first sexual encounter at an early age, and those who reported having a higher number of sexual partners. All these variables were observed in this cohort.
Professor Luisa Villa: Indeed, it was pioneering in our region, demonstrating that women infected with a persistent high-risk HPV are those who may later develop cervical cancer. In other women, the infection is transient, with HPV being cleared within a few months. Only about 10% of women are at risk of developing cervical cancer.
It’s also important to highlight the methodology: from the beginning, we used PCR-based methods, which are the best, most sensitive, and most specific for detecting various types of HPV. We tested for a range of 30 HPV types. This allowed us to define risk profiles: HPV-16 and HPV-18 as high-risk, followed by HPV-31 and HPV-45, and about nine or ten intermediate types. Other types are found in cancer but are not as prevalent. Now, those that pose the greatest risk – “the pestilent ones” as I like to call them – are about ten highly oncogenic types. Our study contributed to this knowledge.
C2PO: What were the main challenges and difficulties in conducting such a complex and long-term study?
Professor Luisa Villa: The biggest challenge was planning. This study – and I say this with pride – was meticulously planned by Professor Eduardo Franco and myself. We thought through every detail, and every question was thoroughly researched. Of course, we had reinforcements; we brought in collaborators and worked with people in Brazil and abroad, but we had a very clear vision of the next steps, depending on the outcomes we encountered. We didn’t just consider one snapshot in time but understood the need to keep the study alive, dynamic, and real for many years. After all, it was a follow-up study, and the natural history of cervical cancer doesn’t happen in months but over years: 10 years, 15 years. So, we also had to keep participants engaged for a long time. High retention rates were crucial to the success of the study, and we had two extremely competent nurses. It’s important to mention this because it serves as motivation for those starting to design studies like this to develop their projects well. If not well planned, it falls apart.
Another challenge is maintaining the drive to generate publications. Sometimes you have people working in the lab, but scientific papers take time to write. You can present your findings at a conference, but research is only recognized and valued through international publications.
Moreover, there is always the difficulty of securing financial support for such studies. We had significant support at the time because Professor Eduardo and I were employees of the Ludwig Institute for Cancer Research, directed at the time by Professor Ricardo Brentani from FMUSP. However, we also sought funding from research institutions like FAPESP and the National Institutes of Health (NIH) in the U.S., and this was crucial to sustaining the research for many years.
C2PO: What was the importance of the Ludwig-McGill Study in the training of researchers?
Professor Luisa Villa: The training of dozens of people was an important outcome of this study. Many Masters and PhDs received training within the Ludwig-McGill cohort and contributed results and publications, as is the case with Professor Laura.
The most intense period was the first 20 years. In the first 10 years, researchers were trained who actively worked on handling samples and following the women. We had students from various backgrounds and collaborations with others. We trained people who later went on to conduct their own cohort studies in different states of Brazil and abroad, such as in Argentina.
Today, the Ludwig-McGill cohort represents a rich source of laboratory results and epidemiological surveys, as well as biological samples stored in a biorepository that continues to generate new publications through new analyses, whether through new laboratory tests or new epidemiological analyses aiming to answer new questions.
C2PO: Can you tell us more about the biorepository?
Professor Laura Sichero: The biorepository is a collection of samples obtained during the participants’ visits. Every time they came for a visit, besides answering an epidemiological questionnaire – covering hygiene, sexual variables, sociodemographic variables, etc. – samples were collected. We collected cervical smears for cytological tests and DNA extraction, performed HPV detection tests, and collected a blood sample.
There were about 2,500 women in the first year, who came every four months, and about 2,000 continued to come every six months for another four years. For each woman’s visit, we have a cervical smear, extracted DNA from the cervical sample, and a blood sample. These are tens of thousands of samples. They must be meticulously cataloged and preserved, as it’s a very rich biorepository.
C2PO: What are the current activities of the study?
Professor Laura Sichero: The study will continue to be what it has always been. The questions it was designed to answer were resolved back then. But today, we acquire new knowledge and formulate new questions. I always say: we finish one project, answer one question, and ten more come up. These new questions go beyond the original project but are what keep the study alive.
We have the biorepository and a vast database, not only with sociodemographic data from the participants but also with cytological analysis data, as well as all the variables assessed: host genetic variants, HPV variants, viral load, serology, among others. All this is stored. This database is very rich and continues to grow with new information, answering more current questions.
Some questions we ask today can be answered without needing additional lab tests. Researchers can use the data that already exists. However, sometimes additional tests are necessary, so we retrieve samples from the biorepository, follow all ethical procedures, and conduct new tests to find answers. Thus, the samples collected years ago continue to generate new work, providing new students with opportunities to develop projects and conduct analyses.
C2PO: Is there any specific question on the horizon to be answered?
Professor Laura Sichero: We are currently researching the latency of infection. That is, if you detect a new HPV infection in a woman, is it a new infection or a reactivation of an old infection? This was a question that didn’t exist at the time, and we have some very relevant publications coming out now using the Ludwig-McGill cohort data.
C2PO: Did the study also play a role in formulating public policies, especially regarding HPV vaccination?
Professor Laura Sichero: Professor Laura Sichero: Studies like the Ludwig-McGill study were relevant for public policy because they increased our understanding of the natural history of the infection at the time. We learned that out of the more than 200 types of HPV described, only a fraction causes cervical cancer in women. Today, the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) recognizes 13 types of HPV as causing cervical cancer.
The research was fundamental for the companies that developed the vaccines to understand which types of HPV the vaccines needed to protect against, with the goal of benefiting the largest number of women. The first vaccines protected against HPV-16 and HPV-18, which are the most prevalent high-risk types. These two types of HPV account for 75% of cervical cancers. The research showed that if we could protect against these two types of HPV, we would be able to protect 75% of women.
Additionally, HPV-16 is the most prevalent type in all HPV-related tumors that are not cervical cancer, so most vulvar, vaginal, penile, oropharyngeal, and anal canal tumors related to HPV are caused by HPV-16. Therefore, the vaccine protects against a range of diseases, although the initial knowledge was generated from cervical cancer, the most common HPV-related tumor worldwide.
Furthermore, the quadrivalent vaccine protects against HPV-6 and HPV-11, two low-risk types responsible for 90% of genital warts.
C2PO: And what are the vaccines like now?
Professor Laura Sichero: Today, we have a nonavalent vaccine that protects not only against the four types of HPV included in the quadrivalent vaccine, but also against five additional high-risk types of HPV that, together with HPV-16 and HPV-18, account for 95% of cervical cancers. However, the nonavalent vaccine is not yet available in our public health system. The vaccine we have available is the quadrivalent, which protects against HPV 6 and 11 (90% of genital warts) and HPV 16 and 18 (75% of cervical cancers).
C2PO: Besides the fact that the SUS (Brazilian public health system) does not use the most up-to-date vaccine, are there any other shortcomings in Brazilian public policy for cervical cancer control?
Professor Luisa Villa: In Brazil, the Papanicolaou (Pap smear) test is still used for screening precancerous cervical lesions. This screening is based on the morphology of exfoliated cells from the cervix, which has moderate sensitivity, about 50% to 60%. Thus, half of the women who undergo the test are not being adequately monitored. It would be more appropriate to use an HPV molecular test-based screening methodology. This implementation is starting to be discussed in Brazil, while many countries around the world are already applying this much more sensitive method in cervical cancer screening.
C2PO: What does the transfer of the role of principal investigator of the study from Professor Luisa Villa to Professor Laura Sichero mean?
Professor Luisa Villa: Professor Eduardo Franco and I have followed this cohort for more than 30 years. But the time is approaching for us to retire. In light of that, we decided to transfer the leadership to Professor Laura Sichero here in Brazil and to Professor Helen Trottier in Canada. This means keeping a study with this dynamic, this value, this wealth of data, and human resources alive. It’s something that some researchers sometimes forget to do. Professor Eduardo and I will continue publishing together, but we have passed on the daily work and the work on new projects, with new students, to these two leaders who have accompanied us throughout the process.
Professor Laura Sichero: And for me, it’s an honor to take on this role because many aspects of my scientific career have been guided by studies conducted in this cohort. I did my master’s on genetic variability analysis of HPV-16 using the samples from the study. During my PhD, even while working on other questions, I continued evaluating these samples. Throughout my career, as new questions emerged in other research on HPV, I continued seeking answers in this cohort. It’s a project that has shaped my entire career. Receiving this great responsibility is very important and gratifying.
C2PO: What makes a mentoring relationship as successful as yours?
Professor Laura Sichero: I started working with Professor Luisa in July 1995, so it’s been almost 30 years of partnership. Our relationship is very harmonious and full of admiration. My admiration for Professor Luisa comes from her being my mentor and role model. She taught me how to conduct research rigorously and ethically, to do the work the right way. And I always had a lot of freedom within her group to propose the topics I wanted to research. So, it really is a successful partnership.
C2PO: Finally, what skills are important for the next generation of researchers?
Professor Laura Sichero: What a student fundamentally needs is a passion for science. The passion for asking questions and pursuing the best ways to answer them. This includes the desire to acquire basic knowledge, the desire to acquire specific knowledge, and the willingness to conduct experiments. I have always enjoyed dedicating myself to working as effectively as possible. I always made a point of informing myself not only about the specific aspects of my research but also about basic knowledge of other areas that might influence my work. All of this knowledge can be incorporated into our projects. I tell my students: do you want to only know about your project, or do you want to be able to discuss science? To be able to discuss science, you need broad training; you must be curious and willing to study and learn new things.