From Cell Populations to Epidemiological Population Studies and Back – Neurofibromatosis Research in 50 Decades
Sirkku Peltonen, MD, PhD, Professor of Dermatology and Allergology, University of Helsinki and Helsinki University Hospital
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This is a life-long story of my journey with NF. My research started in 1985 when I, as a third-year medical student at the University of Turku, Finland, joined the newly formed research group of Dr. Juha Peltonen. He had defended his PhD thesis on connective tissue of neurofibromas a few months earlier. The task he gave me was to write a literature review on the culturing of Schwann cells and set up a culture system in the lab. At that time, writing a review was vastly different from today’s digital age. Internet, Medline, and Pubmed were nonexistent, and the literature was searched using big books called Index Medicus, which listed references of new publications according to keywords. The publications in Index Medicus were always several months or more than one year old, and thus, it was important to read the new journals every week to find fresh publications.
Serendipity kicks off my career.
Neurofibromatosis 1 was then called von Recklinghausen’s disease; the NIH conference, which defined the diagnostic criteria, had not been organized, and the disease-causing gene had yet to be discovered. Cell culture and immunohistochemistry, especially immunofluorescence techniques, were cutting-edge tools in cell biology.
My project to culture cells from rat sciatic nerves was successful but not in the way it was meant, since instead of propagating pure Schwann cell populations, the culture flasks became populated by large flat cells, which we showed to be perineurial cells. The project thus resulted in characterization of perineurial cells in culture (Peltonen et al. Lab Invest 1987).
I had the privilege to attend The First European Symposium on Neurofibromatosis, which was organized in Runnymede Hotel, outside of London, in February 1987. All the most prominent researchers in the field, whose studies I had read for my degree work were attending or gave lectures which fueled my interest in NF research.
Research adventures in the USA: more fuel to NF1 enthusiasm
In the summer of 1987, I got the opportunity to join Juha Peltonen in Philadelphia to work as a research assistant in the laboratory of Professor Jouni Uitto at Thomas Jefferson University. Juha got the Young Investigator Award from the National Neurofibromatosis Foundation (now CTF). The Uitto lab used modern DNA technology and tools on connective tissue research, and we applied them to neurofibromas. Studying the expression of various collagen and laminin genes in cells cultured from cutaneous neurofibromas (Jaakkola et al., J Clin Invest 1989) became an important part of my PhD thesis. The results showed that Schwann cells, perineurial cells, and fibroblasts all contribute to the extracellular matrix of neurofibromas.
Professor Uitto helped us to obtain neurofibromas from his dermatologist colleague, Dr. Mark Lebwohl, at Mount Sinai Hospital. In 1988, I traveled numerous times by train from Philadelphia to Manhattan with a box of ice in a bag and went to see when Dr. Lebwohl operated neurofibromas and waited for him to give me the extra tumor tissue to take back to Philadelphia. I was impressed by Dr Lebwohl’s friendly way of talking with the patients and listening to their stories while operating. There I also saw how much NF1 can shape a person’s life and how huge disease burden cutaneous neurofibromas can cause.
The chromosomal location of the NF1 gene was described in the summer of 1989 in two simultaneous publications. We happened to attend the conference in Washington, D.C., in May 1989, where Professor Francis Collins presented two patients with balanced translocations involving chromosome 17q11.2 and led his research group to the identification of the NF1 gene. Listening to this was an unforgettable moment and great news for the NF1 research community. It raised high hopes that the secrets of the syndrome could be revealed and that a cure could be developed. The enormous size of the gene did not give too much hope for rapid diagnostics, but the gene cloning enabled expression studies at the protein level. These studies revealed the complexity of the expression of the NF1 gene at the mRNA and protein level.
After two years in the USA, it was time to return to Finland, defend PhD thesis (entitled: Perineurial cell: Expression of connective tissue genes in rat sciatic nerve and cutaneous neurofibromas), and graduate from medical school. During my last two years as a student at the University of Turku, I happened to meet two patients with NF1, and on both occasions, the doctor was not familiar with NF and did not have answers to the patient’s questions. I became very aware of the knowledge gap and the need for more education on NF for healthcare professionals.
Becoming a mother, dermatologist, and neurofibromatologist
After graduation and an internship in medicine, we moved to Northern Finland, and I worked as a postdoc in the basic science lab at the Department of Biochemistry for 1,5 years. After returning to Turku, I became the mother of our son Ilmari in 1995 and a daughter Alisa in 1998 while specializing in dermatology. I simultaneously contributed to studies of Juha Peltonen, with one focus being expression of neurofibromin in epidermis and epidermal keratinocytes. Confocal microscope was a great new imaging modality which produced amazingly beautiful and informative digital images on neurofibromin in cells and tissues (for example, Koivunen et al, J Invest Dermatol 2000)
While working as a resident in dermatology, I managed to convince the leadership of the Department of Dermatology at Turku University Hospital that we need an NF clinic. The first patients were seen at the end of 1999. It soon became evident that patients and expertise were scattered across different clinics within the hospital. Over time, an increasing number of new referrals came in, but it was only on the second attempt, 15 years after the clinic’s inception, that we managed to sustain regular multidisciplinary NF meetings. I used carbon dioxide laser every week to remove neurofibromas, which also generated valuable research material. This clinic remained the only NF clinic in Finland for the next 25 years and served patients from other parts of the country as well.
When both parents attended the same conferences, it was often simplest to take the children with us. Both, or at least one of them have joined to Aspen, Venice, Gothenburg, Killarney, Istanbul, Barcelona, Baltimore, and Washington, and of course our own conference. In July 2003, we organized the tenth European NF Conference in Turku, Finland, and we were happy to host about 150 congress delegates, both scientists and lay group members, for three sunny days in our hometown. The program contained topics such as NF2 and NF2 mutation analyses, bone, cancer, and several basic science aspects in cell biology sessions. The combined clinical session for NF1 and NF2 included topics that are still relevant and not totally solved, such as the correlation of MPNST with deeper neurofibromas or growth dynamics of cutaneous neurofibromas.
Millenium: trying to follow the advances in DNA technology
In the 2000s we saw a rapid advance in technology: DNA arrays and NGS sequencing. The presentations at the NF conferences in the USA contained mostly molecular studies, including numerous animal models and mutation analyses. I presented posters on gene expression studies and intercellular junctions, but the most interesting poster, as estimated by a number of personal contacts at the poster exhibition, was the one in Baltimore in 2010 entitled: Removal of cutaneous neurofibromas with carbon dioxide laser.
Using modern technologies became expensive, and when working in a small town with a limited budget and a small number of patient samples, we were somewhat overrun by technologies and results from larger labs. However, we went on using the old immunohistochemistry to study human tissue, for example intercellular junctions in skin and neurofibromas. We finally found a marker for perineurial cells: tight junction component claudin-1 was expressed by perineurial cells both in nerves and in neurofibromas (Pummi et al. J Histochem Cytochem 2001 and 2004). Using immunolabeling, we also dug into the “roots” of neurofibromas and demonstrated nascent neurofibromas residing in association with hair roots (Jouhilahti et al. Am J Pathol 2011)
Lessons from epidemiology: NF1 has profound implications on many aspects of life
The 2010s was marked by the emergence of clinical studies carried out in the USA to find medical treatments for NF1 tumors. Many sessions in CTF meetings reported unsuccessful attempts. Finally, the MEK inhibitors proved to be functional, and children with symptomatic plexiform tumors can now have an opportunity for treatment. Alongside clinical studies, the CTF conferences have grown and included more clinically oriented sessions, which has been a pleasure for a clinician scientist.
Since 2010, I have mainly worked on epidemiological studies on NF1. We collected a cohort of about 1500 patients, verified their diagnoses in the medical records, collected their first-degree relatives from the Population Register, and followed their health and life in the Finnish health, birth, death, education, and income registries. Reading hundreds of patient records taught me how many different life stories the patients have. Sitting in archives also opened viewpoints to the expertise – or merely knowledge gaps of health care professionals. The population study is still ongoing, and to date, it has produced about 15 publications on various aspects of the health and life of patients with NF1. The most important to date has been the analysis of cancer incidence and mortality (Uusitalo et al. J Clin Oncol 2016, Peltonen et al. Int J Cancer 2019), which demonstrated the extremely high risks for nervous system malignancies and confirmed the increased breast cancer risk in women. This is essential knowledge for the clinical management of patients. Fruitful collaboration with health economists has enabled us to demonstrate how big an impact NF1 has on educational level and, furthermore, performance in the labor market. Unfortunately, having NF1 leads to markedly lower lifetime income compared to the control population, and NF1 may be seen as a source of hereditary economic inequality (Johansson et al. Genet Med 2022). The register studies are still ongoing and will draw a sharper picture of the lifespan of patients with NF.
The 2010s have also shown an emergency of immunology in the field of tumor biology. In 2017, we applied and were granted research funding from the Neurofibromatosis Therapeutic Acceleration Program (NTAP) for a project on tumor immune microenvironment in cutaneous neurofibromas. I was back carrying out cell population studies, which analyzed mast cells (Kallionpää et al. Dermatology 2022) and T cell and macrophage populations of neurofibromas (Kallionpää et al. Lab. Invest 2023). I believe that studying NF further from the immunology point of view will be necessary.
Strengthening the networks
2010s can also be called a decade of networking. The revision of NF diagnostic criteria was an exciting and important project, and I was very pleased to get an invitation to participate in the dermatology working group. Although dermatologists often diagnose and treat NF1, we are all too few in the NF1 field. (You can blame me if you think that it was a mistake to leave out nevus anemicus and juvenile xanthogranulomas from the diagnostic criteria.)
Networking became more official when the EU Commission established European Reference Networks (ERNs) for rare diseases in 2016. I have been happy to be involved in ERN for Genetic tumor risk syndromes, ERN GENTURIS, from the very beginning. Applying for membership and holding it through evaluations and reporting is a challenge, but it has deepened the collaboration of European clinicians working with NF patients. An expert group for NF1 guideline was established and started working in March 2020. The group, which had members from seven countries, gathered at numerous Zoom meetings and did a lot of homework through the pandemic. I believe that we all are proud of the result, which can be found at https://www.genturis.eu/l=eng/Guidelines-and-pathways/Clinical-practice-guidelines/Neurofibromatosis-type-1-guideline.html (Carton et al. EClinicalMedicine 2023).
In 2020, I was appointed as Professor of Dermatology at the University of Gothenburg, Sweden, and doctor-in-chief at the Department of Dermatology and Venereology at Sahlgrenska University Hospital. Like Finland, Sweden has no official NF centers, and the specialists taking care of the patients are scattered throughout the hospital. I started the NF clinic in Dermatology and, in collaboration with the Center of Rare Diagnoses, again formed a network of specialists from different clinics.
While working in Sweden, NTAP contacted me with a suggestion to participate in a study to test high-intensity focused ultrasound techniques on cutaneous neurofibromas. NTAP team, a group of researchers from Copenhagen, Denmark, and some of my Swedish colleagues from Gothenburg teamed together and carried out a study where we treated 147 cutaneous neurofibromas. The study resulted in quite promising results (Peltonen et al. JEADV Clinical 2024). I hope the Danish colleagues will continue to improve the technique to the most feasible method for cutaneous neurofibromas.
Life took me back to Finland and since April 2023 I have worked as a Professor of Dermatology and Allergology in University of Helsinki and a specialist in the Helsinki University Hospital – and started the third NF clinic in my career. Networking with colleagues in Helsinki is still ongoing.
Constant need for NF1 education
My mission as a teacher has been to increase knowledge and awareness among healthcare professionals regarding NF. As a dermatologist, I have always represented a small minority among NF scholars, and a neurofibromatologist among dermatologists belongs to a marginal. Luckily, I have had several opportunities to educate dermatologists on NF in European, Scandinavian, and national meetings, while my husband Juha has mostly been invited to represent our team in numerous NF meetings.
The other mission I have had since my first contact with patients almost 40 years ago has been to educate patients on their disease. This is a weekly challenge when running an NF clinic. I have promoted two patient information booklets in Finnish: one for adults and one for children and authored the Swedish NF1 information to the official web page of rare diseases https://www.socialstyrelsen.se/kunskapsstod-och-regler/omraden/sallsynta-halsotillstand/neurofibromatos-typ-1/. The latter text was commented several times by a review group of health care professionals and lay persons. The lesson I learned was that lay persons and many professionals are not ready yet to accept the high cancer risk related to NF1.
As I reflect on the early days of my career, I am humbled by the progress made since then. I am grateful for the opportunity to work with many great scientists and people. They are too numerous to list, and therefore, I purposely do not mention any by name. NF research has evolved, diagnostic criteria have sharpened, and groundbreaking discoveries continue to reshape our understanding of this complex disorder. I hope to be able to see more and more hope in the future.
