Trial Information

Analysis of Circulating Tumor DNA in Plasma of Neurofibromatosis Type 1 Patients With MPNSTs Using Microarray CGH

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 oncogene. Clinically, NF1 patients have
café-au-lait maculae, freckling, Lisch nodules and neurofibromas. Cognitive problems, bone
lesions and optic pathway gliomas are also common in these patients.

Neurofibromas are benign neoplasms of the peripheral nerve sheath, which can appear anywhere
in the body of NF1 patients. 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, a neurofibroma will be formed. There are 3 types of
neurofibromas: cutaneous, subcutaneous and plexiform. Cutaneous 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. Subcutaneous
neurofibromas are discrete nodules located on peripheral nerves and the timing of their
origin is not known, but they are probably also prenatal in origin.

Every NF1 patient has a life time risk of 8 to 13% to develop a malignant peripheral nerve
sheath tumor (MPNST). This risk is even twice as high for a subpopulation of NF1 patients,
namely patients with an NF1 microdeletion. MPNSTs are difficult to diagnose in early phase
because of the large number of tumors and diverse locations often seen in these patients.
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 and the fact that the tumors are frequently already metastasized at diagnosis. The
five-year survival of patients with MPNSTs is less than 25%. For MPNSTs, like for other
cancers, the extent and the spread of the disease at time of diagnosis is an important
factor in determining treatment outcome. In this regard, tumor markers have been developed
to screen for tumors, but only a subset of cancers secrete specific proteins that can be
used as a marker. An increasing interest grew in new markers like circulating nucleic acids.
The detection of cell-free circulating nucleic acids in plasma, serum and other body fluids
of healthy and diseased individuals opened up the possibility to diagnose and monitor the
disease, which might also be applicable to NF1 patients with MPNSTs. The presence of
extracellular circulating nucleic acids in the blood of healthy and sick persons was first
described by Mandel and Métais in 1948. In 1977 the group of Leon was the first to quantify
cell-free DNA in serum. They found that cancer patients tended to have elevated levels of
circulating DNA compared to healthy control individuals, and that the amount of DNA was even
bigger in patients with metastases compared to localized disease, but nothing was known
about the origin of the DNA. Significant progress in plasma/serum DNA research was made,
when the presence of tumor-associated microsatellite alterations was reported in the plasma
and serum of cancer patients. According to these findings, it has been suggested that the
circulating DNA could have potentials for the diagnosis and prognosis of malignancies.
Still, the mechanism of DNA release is not fully clarified, and different hypotheses have
been suggested. A first hypothesis that the circulating DNA originates from DNA leakage due
to necrosis or apoptosis of tumor cells, became controversial when it was reported that the
DNA level decreased after radiotherapy. Another hypothesis about micrometastases was also
rejected because the number of cells in plasma didn't match the amount of circulating DNA. A
third hypothesis is the spontaneous and active release of tumoral DNA in the blood by
proliferating cells. However there is no convincing explanation for the latter mechanism.

Research protocol A first part of the study will be the optimization of the technique of DNA
extraction from plasma. Different DNA extraction methods described in the literature will be
tested on samples collected in cooperation with Prof. Dr. Patrick Schöffski (UZLeuven). When
blood is taken from cancer patients for routine testing, an additional 5 mL (in EDTA tube)
will be available for our experiments. Blood samples will be collected within a few hours
after sampling and processed within 24 hours in our lab (lab for neurofibromatosis
research). The blood samples will be centrifuged at 1600 rpm for 10 minutes. The supernatant
plasma will be collected, without disturbing the buffy coat, and re-centrifuged at 13000 rpm
for 10 minutes to remove particles. The supernatant plasma will be aliquoted into 0.5 mL
volumes. The samples will be stored at -80°C until further use. Per patient, different
aliquots will be extracted using different methods, like phenol-chloroform extraction and
commercial kits, according to the literature. Genomic reference DNA will be extracted from
the buffy coat using phenol-chloroform extraction. Depending on the type of DNA extraction,
we expect differences in quantity and quality of the DNA. The method with the highest yield
and best DNA quality will be chosen to extract DNA from plasma of NF1 patients with MPNSTs.

Second part of this work will be the optimization of the microarray experiment using plasma
DNA. Plasma samples from NF1 patients with MPNSTs will be collected in collaboration with
the group of Victor Mautner from the Eppendorf University in Germany. Direct processing of
blood samples will be done in Germany and frozen buffy coats and plasma aliquots will be
sent to Leuven in an encoded way. Together with the buffy coats and plasma, frozen tissue of
the patients MPNST will be sent as well. DNA from the tumor will be extracted using
phenol-chloroform extraction. Microarray CGH will be performed on both the tumor and the
plasma DNA with buffy coat DNA as reference to exclude copy number variable regions from the
list of copy number changes. The array profiles of the plasma DNA will be checked for the
tumor-related copy number changes. In that way, the array protocol can be further optimized
in order to detect tumor specific copy number abnormalities in the cell-free DNA from
plasma.

This study is performed by Eline Beert, PhD student in the lab for neurofibromatosis
research, and Radost Ratcheva, a master thesis student in the lab of neurofibromatosis
research, as part of achieving the degree of 'Doctor in Biomedical Sciences' and 'Master in
Biomedical Sciences', respectively, with Prof. Dr. Eric Legius as promotor and main
researcher. Only the Katholieke Universiteit Leuven and the UZ Leuven, 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 of it
will be reported to the patients.

The blood samples of the UZLeuven cancer patients will only be used to optimize the DNA
extraction procedure, the extracted DNA will be quantified but it will not be further
analyzed and will be destroyed afterwards.