Running Title: Adrenomedullin as an NF1 serum biomarker

and a secreted protein database we identified upregulated transcripts in human plexiform neurofibromas (pNF) and MPNSTs. Further investigation revealed that adrenomedullin (ADM) was found in the serum of NF1 patients at higher levels than controls and was also found to higher levels in the sera of patients with MPNST compared to those with pNF, suggesting that ADM may be a serum biomarker for malignant transformation. If validated in a larger study, serum levels of ADM could serve as an additional tool in the diagnostic repertoire of the physician when attempting to determine if a plexiform neurofibroma has undergone malignant transformation. Abstract Purpose: Plexiform neurofibromas (pNF) are Schwann cell tumors found in a third of individuals with neurofibromatosis type 1. pNF can undergo transformation to malignant peripheral nerve sheath tumors (MPNST). There are no identified serum biomarkers of plexiform neurofibroma tumor burden or transformation to MPNST. Serum biomarkers would be useful to verify NF1 diagnosis, monitor tumor burden and/or detect transformation. Experimental Design: We utilized microarray gene expression analysis to define 92 genes that encode putative secreted proteins in neurofibroma Schwann cells, neurofibromas and MPNST. We validated differential expression by QRT-PCR, Western blotting and ELISA assays in cell conditioned medium and control and NF1 patient sera. Results: Of thirteen candidate genes evaluated, only adrenomedullin was confirmed as differentially expressed and elevated in serum of NF1 patients. Adrenomedullin protein concentration was further elevated in serum of a small sampling of NF1 patients with MPNST. MPNST cell conditioned media, containing adrenomedullin and hepatocyte growth factor, stimulated MPNST migration and endothelial cell proliferation. Conclusions: Thus, microarray analysis identifies potential serum biomarkers for disease, and adrenomedullin is a serum biomarker of NF1. Adrenomedullin serum levels do not seem to correlate with presence of plexiform neurofibromas but may be a biomarker of transformation to MPNST. After 18 hrs the plates were stained using Calcien AM (8 ug/ml) and two non-overlapping fields per well were photographed using a fluorescent microscope, accounting for >80% of the area of each filter. Three wells were analyzed per experimental condition. Three independent experiments were conducted and data from a representative experiment is shown. The area of tube formation was then quantified using Image J software. The present study describes integration of NF1 transcriptional data and human proteins known or predicted to be secreted. From a list of 92 putative secreted NF1 biomarkers, we identified two candidates: ADM and HGF. ADM and HGF mRNA differential expression were validated by qPCR. Both proteins are secreted in serum-free conditioned media from sporadic and NF1-associated MPNST cell lines. We demonstrate that ADM is a serum biomarker of NF1; NF1 patients with and without pNF, and with MPNST, have significantly increased serum concentrations of ADM compared to controls. We also show that ADM has cell autonomous effects on MPNST migration. While thirteen candidates were initially investigated, another 79 candidates have yet to be validated. Further investigation of these candidates using serum-free conditioned media and the sera of patients with pNF and MPNST as described above could lead to additional biomarkers and potential therapeutic targets