Analysis of Peripheral Nerve Sheath Tumors in Neurofibromatosis Type 1 Patients
Introduction Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder occuring in 1
out of 3500 living newborns. The disease is caused by heterozygous mutations of the NF1
gene, located on chromosome 17q11.2. The NF1 gene encodes the tumor suppressor
neurofibromin, a negative regulator of the RAS/MAPK pathway. In approximately 95% of
patients, the heterozygous mutation is intragenic. The remaining 5% of patients are carrier
of a microdeletion, mostly the type 1 deletion, in which apart from the NF1 gene, 14
additional protein coding genes are located.
Clinically, NF1 patients have café-au-lait maculae, freckling, Lisch nodules and
neurofibromas. Also cognitive problems, bone lesions and optic pathway gliomas are common in
Neurofibromas are benign neoplasms of the peripheral nerve sheath, which appear everywhere
on the body of almost every NF1 patient. Neurofibromas are composed of different cell types
like Schwann cells, fibroblasts, mast cells and perineurial cells. If in Schwann cells of
NF1 patients the wild-type NF1 allele is inactivated in a heterozygous microenvironment, a
neurofibroma will be formed. There are 3 types of neurofibromas: cutaneous, subcutaneous and
plexiform. Cutaneous and subcutaneous neurofibromas appear during adolescence as isolated
nodules in or under the skin, respectively. Plexiform neurofibromas are congenital and can
spread along a large segment of a peripheral nerve. NF1 microdeletion patients have in
general more neurofibromas and they appear at a younger age.
Every NF1 patient has a life time risk of 8 to 13% to develop a malignant peripheral nerve
sheath tumor (MPNST) out of a pre-existing (plexiform) neurofibroma. This risk is even twice
as high for NF1 microdeletion patients compared to patients with an intragenic mutation.
MPNSTs are difficult to diagnose in early phase because of the large number of tumors and
diverse locations. These tumors infiltrate easily in surrounding tissue and frequently give
rise to metastases. At this moment, the only available treatment is surgical removal of
these MPNSTs. Complete resection is often difficult due to the size of the tumors and the
location on important nerves. The five-year survival of patients with MPNSTs is less than
Research protocol All NF1 patients are linked to the UZ Leuven. Their follow-up is done by
Prof. Dr. Eric Legius. When the patient is suspected of having a MPNST, the patient is
referred to the surgeon of the clinical oncology department, respectively. Also
neurofibromas are removed when they are causing problems for the patient. As part of the
different tumor care programs in our hospital, a tumor piece is sent from pathology to the
centre for human genetics for routine genetical analysis, like karyotyping. In the lab for
neurofibromatosis research, the rest material is frozen as tumor pieces and as dissociated
cells for research purposes. The samples are stored in a database system that is accessible
only for the neurofibromatosis research group. In cooperation with the group of Victor
Mautner and Karen Cichowski, the tumor database was further completed with NF1 tumor
material and cell lines. The latter samples were coded and could be used as part of our
Experimental approach In literature, little is known about the development of a MPNST out of
a neurofibroma. In this study we want to gain insight into this process, which can in the
end lead to a better diagnosis and treatment of NF1 patients with MPNSTs. For this purpose,
only the frozen rest material of neurofibromas and MPNSTs will be used.
At first, DNA will be extracted from the frozen tumor pieces (neurofibromas and MPNSTs).
Together with blood DNA (also rest material), the tumor DNA will be screened with microarray
genomic hybridization. This technique allows us to check for chromosomal abnormalities, like
amplifications and deletions, with a much higher resolution than traditional cytogenetics.
In that way, we can look for recurrent alterations within a certain tumor type and for
similarities and differences between benign and malignant tumors.
Secondly, the group of MPNSTs will be subdivided in tumors derived from NF1 microdeletion
patients and tumors from patients with an intragenic mutation. Recurrent chromosomal gains
and losses will be compared between the 2 groups. This will show us if the mechanism of
tumor development and progression is different in NF1 microdeletion patients. In cooperation
with the group of André Bernards and Karen Cichowski, we will try to elucidate the role of
the NF1 microdeletion in the development of neurofibromas. Our group will collect all tumor
samples and grow Schwann cells and fibroblasts out of these tumors. The frozen vials with
tumor pieces and cultured cells will be encoded and provided to these labs as part of our
Subsequently, the DNA derived from 10 different tumors (neurofibromas and MPNSTs) and
matching blood DNA will be analyzed by 'full exome sequencing' to check for mutations in all
In collaboration with the group of Yuan Zhu, tumor samples will be sent and retrieved in an
This study is performed by Eline Beert, PhD student in the lab for neurofibromatosis
research, as part of achieving the degree of 'Doctor in Biomedical Sciences', with Prof. Dr.
Eric Legius as promotor and main researcher. Only the Catholic University of Leuven and
other foreign universities, but no commercial partners (like companies) will participate in
this study. The results obtained from the described experiments have to be considered as
pure scientific information and none will be reported to the patients.
Observational Model: Cohort, Time Perspective: Retrospective
Eric Legius, MD PhD
Katholieke Universiteit Leuven
Belgium: Ethics Committee