NF1 and Cutaneous Neurofibroma

Cutaneous neurofibromas (cNF) are an unmet need within the complex syndrome of NF1 and patients with NF1 often identify these tumors as their greatest burden. As part of our commitment to helping patients with NF1, the Neurofibromatosis Therapy Acceleration Program (NTAP) has taken on the targeting of Cutaneous Neurofibromas as part of its overall mission. To launch our efforts in this area, NTAP hosted a summit on cutaneous neurofibromas towards the end of 2016 to create a community of invested thinkers with the resources and time necessary to explore key questions pertaining to developing effective therapeutics for cNF in people with NF1. The intimate and focused 2 day summit was an interactive meeting with the major goal of identifying the key questions for understanding and treating cNF, such that post-meeting NTAP could launch requests for applications (RFAs) aimed at addressing those questions in the next 3 years. Experts, with and without explicit experience with NF1 or cNF, from disciplines across biology, chemistry, dermatology, surgery, skin cancer, regenerative medicine/tissue repair, wound healing, genetics, and immunology, representing the ranks of academia, industry, and government agencies met to discuss at an in-depth level various scientific topics associated with disease pathogenesis and treatment within the following major themes:

• Disease Initiation, Progression, Development
• Therapies Development
• Clinical Trials Design, Optimization, Development

From these efforts, a first request for applications (RFAs) was announced in 2017 to spur the highest priority research for cutaneous neurofibroma.  From this RFA announcement, 9 projects were identified and activated in 2018.

NTAP cutaneous neurofibroma summit scientific partners

The persons listed below (alphabetically) were presenters at the summit, and are vested scientific partners with NTAP in advancing our efforts in targeting cutaneous neurofibroma. A core value of NTAP is collaboration, and this group will work with us to seek partners to help us in our shared cause – to accelerate the development of medicines for patients with cutaneous neurofibroma.

Richard ‘Rox’ Anderson, MD (Harvard, Massachusetts General Hospital)
Jaishri Blakeley, MD (NTAP, Johns Hopkins)
Isaac Brownell, MD, PhD (National Cancer Institute)
Denise Casey, MD (Food and Drug Administration)
Kurt Jarnagin, PhD (Anacor/Pfizer)
Hon-Sum Ko, MD (Food and Drug Administration)
Bruce Korf, MD, PhD (U. Alabama)
Robert Lavker, PhD (Northwestern)
Lu Le MD, PhD (U. Texas, Southwestern)
James Lee, MD (Roivant Pharmaceuticals)
Eric Legius, MD, PhD (U. Leuven)
Scott Plotkin, MD, PhD (Harvard, Massachusetts General Hospital)
Vincent ‘Vic’ Riccardi, MD, MBA (The NF Institute)
Sharad K. Verma, PhD (NTAP, Johns Hopkins)
Hubert Weinberg, MD (Private Practice, and Mt. Sinai)
Brigitte Widemann, MD (National Cancer Institute)
Pierre Wolkenstein, MD, PhD (Paris Est University)

CUTANEOUS NEUROFIBROMA INITIATIVE PROJECTS (initiated 2018)

“Leveraging human iPSCs to determine the impact of patient-derived NF1 gene mutations on peripheral sensory neuron-driven Schwann cell growth”

David Gutmann, MD, PhD (Washington University, St. Louis, Missouri)

Dr. Gutmann and co-workers will investigate the impact and potential mechanism of peripheral neurons on NF1-deficient Schwann cell growth, with respect to : 1) Generating and characterizing sensory neurons from selected iPSCs, 2) Showing that sensory neurons with or without NF1 deletion have differential effects on SC which are NF1 expressing and non-expressing, and 3) Evaluating neurofibromin expression over multiple time points to determine if there are differential effects of neurofibromin on SC of various NF1 backgrounds over time.


“Modeling cNF with human SC via tunable and reversible control of NF1 protein”

Gabsang Lee, DVM, PhD (Johns Hopkins University, Baltimore, Maryland)

Dr. Lee and co-workers will seek to understand the effect that neurofibromin protein expression levels at specific stages of Schwann cell development have on disease pathogenesis.  The team will investigate the role of NF1 at different stages of neural crest/Schwann cell development by utilizing “isogenically paired” NF1+/+, NF1+/- and NF1-/- pluripotent stem cells, and SMASh (small molecule-assisted shutoff) tag methodology, which allows the tunable and reversible control protein production/degradation (such as NF1 protein).


“Characterization and dissection of cNF Schwann cell-fibroblast interactions” 

Edu Serra, PhD, (The Institute for Health Science Research Germans Trias i Pujol (IGTP) Barcelona, Spain)

Dr. Serra and co-workers will explore in detail the role of heterotypic interactions between SC and fibroblasts on cNF pathogenesis.  They will evaluate heterotypic interactions between SC and fibroblasts in cNFs to specifically identify the gene expression profiles produced, and dissect from the expression profiles which part is expressed by SC and which part by fibroblasts.


“Use of the mouse model of cNFs for identification of cells of origin and drug screening studies”

Piotr Topilko, PhD (INSERM, Paris, France)

The team led by Dr. Topilko will seek to utilize their newly developed genetically engineered mouse model of cNF based on Prss56Cre, NF1fl/-, R26tdTom to: 1) evaluate stromal changes in the progression from micro cNFs to cNFs, 2) identify the derivatives of neural-crest-derived basal cap cells that give rise to cNFs, and 3) test the efficacy of a MEK inhibitor (Selumetinib) in inhibiting or preventing formation of cNFs in the mouse model.


“Cutaneous Neurofibroma: Models, Biology and Translation”

Lu Le, MD, PhD (University of Texas, Southwestern Medical Center, Dallas, Texas)

Dr. Le and co-workers will utilize their genetically engineered mouse model of cNF based on Hoxb7-Cre; Nf1flox/flox to: 1) Identify the cNF cell of origin (from human iPSC) with focus on establishing the presence and location of HoxB7 in human cells and its characterization as a cell of origin marker, and 2) Conduct mouse xenograft studies involving murine derived SKPs with respect to the transplant of in vitro skin reconstructs into hormone-primed nude mice in vivo.


“Genetic Studies of Neurofibromatosis”

Peggy Wallace, PhD (University of Florida, Gainesville, Florida)

Dr. Wallace and co-workers will investigate the genetic factors underlying the pathogenesis of cNFs with respect to: 1) Creation of semi-immortalized SC of various NF1 backgrounds (NF +/- vs. NF -/-) from heterogenous human cNFs, 2) Conduct of single cell authentication and exome RNA sequencing in the semi-immortalized Schwann cell lines  and analysis to compare to results previously obtained from semi-immortalized SC from plexiform neurofibromas, and 3) Evaluating intact cNFs (from primary samples) to ascertain an RNA signature from the SC in the setting of an intact microenvironment, with comparison back to the isolated primary and semi-immortalized SC.


“Microenvironmental targets of cNFs: T-cells and mast cells as tumor contributors”

Juha Peltonen, MD, PhD, and Sirkku Peltonen, MD (University of Tirkku, Tirkku, Finland),

Drs. Peltonen and co-workers will investigate the roles and effects of T-cells and mast cells as microenvironment components in promoting tumorigenesis with respect to: 1) characterization and quantification of T cells residing in cNFs, 2) evaluation of the clonality of T cells, 3) characterization and quantification of cNF mast cells by measuring relative abundancies of mast cell types, 4) studies on expression of NGF and receptors for progesterone and estrogen in cNF mast cells, and 5) evaluation of T-cell-Schwann cell interactions and mast cell-Schwann cell interactions by mass spectrometry.


 “A 3D cNF Model for Automated High-Throughput Drug Screenings”

Alice Soragni, PhD, (University of California, Los Angeles, Los Angeles, California)

Dr. Soragni and co-workers will seek to develop and characterize a miniaturized 3D preclinical model (i.e. a ring organoid) for human cNF and then to validate the organoids by determining whether histopathology, transcriptional profile and pathway alterations of the 3D cNF organoids reflect the basic physiology of the tumor.


“Deconstruction and Reconstitution of NF1 cNF”

Ray Mattingly, PhD, (Wayne State University, Detroit, Michigan)

Dr. Mattingly and co-workers will deconstruct human cNFs using single cell transcriptomics (via scRNA-Seq) to define cell populations involved in the tumor, and then develop pathomimetic avatars (based on those sub-populations) from which  3D co-culture models of the tumors will be developed for preclinical screening.

Cutaneous Neurofibroma Supplement Paper summaries

The Clinical Definition of cNF

Since characteristics of cNF and other neurofibromas can overlap, there has been uncertainty about how to define and classify cNF. Various classification schemes have been proposed, but the lack of consensus has hindered consistent communication among clinicians and scientists, slowing progress on the development of new therapeutics. In the second paper of the supplement, titled ‘Cutaneous Neurofibromas: Current Clinical and Pathologic Issues,’ these challenges are discussed. The paper presents the existing classification schemes for the various forms of neurofibromas and the learnings from two multidisciplinary meetings. The experts in these meetings reviewed the core features of cNF (including clinical and histological features, and data about effects of cNF on quality of life) and they propose necessary elements for a modern classification scheme for cNF. Taking into consideration the learnings from previous efforts, an initiative is now underway to create a commonly accepted classification scheme validated in a prospective study with blinded review by dermatopathologists, neuropathologists and NF1 clinicians that accurately describes each tumor type by its clinical classification, clinical appearance, pathological, and molecular and histological features. By having such a classification scheme in place, researchers, clinicians, drug makers, and regulators will be able to communicate more consistently, thereby facilitating research and discovery aimed at developing tumor-specific management strategies.

The Biology of cNF

Summit participants agreed that the current limited knowledge of the underlying mechanistic, structural, and genetic factors responsible for the formation of cNF represents a major hurdle to understanding the progress of the disease and ultimately to being able to develop effective treatments. To identify knowledge gaps and define future research priorities, the authors undertook a comprehensive examination of published (and unpublished) results from which five key areas of research priority were identified. As described in the third paper of the supplement, titled The Biology of Cutaneous Neurofibromas,’ these are: (1) identifying the human cells of origin for cNF and understanding how these cells influence and drive cNF initiation and progress, (2) understanding how both nerve and tumor microenvironment contribute to cNF development, (3) identifying and elucidating the specific genetic and molecular factors that underlie cNF initiation and progression, including factors that contribute to tumor size and number, (4) generating preclinical model systems to elucidate disease biology and enable preclinical therapeutic testing and (5) understanding how age and sex hormones influence tumor growth. These research topics provided the basis for a first request for applications (RFA) sponsored by NTAP in 2017, from which nine projects were launched. These coordinated projects are a critical early step towards answering key questions that will facilitate the development of therapeutics for these tumors.

Therapeutic Development

Current clinical management of cNF involves surveillance or some form of procedure-based treatment, but lacks a therapeutic (drug-based) approach. To overcome barriers to new treatment development and focus resources on options most likely to succeed, a working group of experts reviewed existing methods, outcomes, patient characteristics, and drug trials for cNF. Insights into current procedural approaches, endpoints applied to assess biologic effect and clinical benefit, and specific factors to consider when developing a drug-based therapy are described in the fourth paper of the supplement, titled ‘Considerations for Development of Therapies for Cutaneous Neurofibromas.’ A key finding is that, while multiple procedures are available for the removal of cNF and each provides treatment options for patients, there were several limitations to the trials that evaluated these approaches. These included a lack of detailed enrollment criteria, distinction among the different types of cNF evaluated, comparisons of treated versus untreated areas, duration of follow-up, and standardized endpoints. Additional, unaddressed challenges include scarring, pain, risk of infection, and the cost and availability of the interventions.  As such, no single approach has been identified as optimal, owing to a lack of prospective data about short- and long-term adverse events or the efficacy of treatments.  Beyond procedure-based techniques, there have been a few interventional drug trials for cNF, but outcomes were highly variable. As cNF is a disease of a nonfatal nature with relatively limited patient numbers, it was concluded that for any product to have a higher likelihood of acceptance, it will have to demonstrate an effect that is clinically meaningful, have a safety profile conducive to long-term dosing, and have a low manufacturing cost. In consideration of these factors, four specific elements were identified as being critical to incorporate into a cNF drug development path : (1) safety, (2) anatomic distribution of cNF, (3) numbers of tumors to be treated, and (4) route of administration. As the number, size, and distribution of tumors is so variable among patients, different groups of patients will require different drug development paths and thus provide opportunities for multiple therapeutic approaches.

Clinical Trial Design

The fifth and final paper of the supplement, titled ‘Clinical Trial Design for Cutaneous Neurofibromas,’ describes recommendations to consider for cNF clinical trials. The priority areas presented include: (1) assessment of existing methods for the measurement and quantification of cNF, (2) advances in technological methods and outcome tools utilized in other skin diseases that may be applicable to cNF studies, and (3) consideration of clinical factors that may influence or confound clinical trial outcomes, including sex and age. To guide clinical trial design, a sound understanding of the natural history of cNF is required. The authors report that two ongoing studies of this kind have led to some important insights, but go on to identify six areas that should be addressed in future prospective studies: (1) the rate of appearance and development of cNF in children and young adults, (2) the growth rates of new versus mature tumors, (3) the influence of hormones and other growth factors on tumors, (4) the rate of spontaneous tumor shrinkage, (5) the identification of biomarkers, and (6) an evaluation of tools used to count and measure tumors. The group agreed that all cNF clinical trials should monitor tumor size, tumor number and patient-reported outcomes to assess efficacy but it emphasized that consensus must first be reached on best methods for monitoring each type of outcome. To this end, the authors detail the tools available, including several developed for other skin diseases. In addition to studying the utility of each technology as applied to cNF, they recommend the creation of a global assessment scale to consistently capture therapeutic effects on patients’ quality of life.