Supplementary table S1: Clinical and histological characteristics of the 10 patients with MPNST

Enlargement Neurological Tumor Tumor Histoprognostic Patient N° Sex/Age Pain of mass signs location size (cm) grade

MPNST-1 M/31 - + - Face 15 III MPNST-2 M/24 + + + Abdomen 20 III MPNST-3 F/19 + + - Lower limb 27 III MPNST-4 M/34 + + + Lumbar spine 7 III MPNST-5 M/18 - + - Head 15 III MPNST-6 F/18 + - - Upper limb 20 III MPNST-7 M/53 + + + Upper limb 13 III MPNST-8 F/24 + + + Lumbar spine 7 I MPNST-9 M/35 + + + Abdomen 20 III MPNST-10 M/33 + + - Face 10 III

F indicates female; M, male Plus sign indicates present; minus sign, absent

Supplementary table S2: Oligonucleotide primer sequences

PCR Genes Oligonucleotide Sequence product size (bp)

TNR Upper primer 5' – AAG AAA GTG CTG CCA CAG GAC AA - 3' 88

Lower primer 5' – CAG ATG CAG CCA CAG GAC TCA A - 3'

TNN Upper primer 5' – AGC ATC TAC TTG CCA CCA CAG ACC - 3' 80

Lower primer 5' – CAC GTC AAG GGA GTT CTC AGT CTC A - 3'

TNXB Upper primer 5' – GCT TCC TCA CCA CGG TTC CT - 3' 67

Lower primer 5' – GCG AAT CCC TCG GTC AAG TT - 3'

TNC Upper primer 5' – GAG GGT GAC CAC CAC ACG CTT - 3' 73

Lower primer 5' – CAA GGC AGT GGT GTC TGT GAC ATC - 3'

TNC-ft Upper primer 5' – GTG TGA AGG CAT CCA CTG CCA T - 3' 86

Lower primer 5' – CCT CCA GCT GAC AGT AGC CGA AT - 3'

TNC-v-A1 Upper primer 5' – GAG CTG GAA AAC CTC ACC GTG AC - 3' 60

Lower primer 5' – GGT CCA GTT GAG TCT GAG GCC AT - 3' Formatted: French (France)

TNC-v-A2 Upper primer 5' – CCC AGG AGG ACT GAG GTC CA- 3' 97

Lower primer 5' – GGT TGT GCT GAA GTC CTG AGT GA - 3'

TNC-v-A3 Upper primer 5' – AGG TTA GCT GGG ATG CTC TCA GAC T - 3' 87

Lower primer 5' – GGT CAG CCT CCT GGA CCT GAA TA - 3'

TNC-v-A4 Upper primer 5' – CAA CTG GAC CGC AGC TGA CAA T - 3' 77

Lower primer 5' – GGG CTG CCT CCA CTT TGT TGA - 3'

TNC-v-B Upper primer 5' – TGC TGA ACG AAC TGC CCA TAT CT - 3' 111

Lower primer 5' – GTG GCT GTG GCA CTG ATG GTT - 3'

TNC-v-AD2 Upper primer 5' – TGA CAT AAC TCC CGA GAG CTT CAA - 3' 111

Lower primer 5' – TAT TCC ACA GTC TCC AGC AAC CTA TT - 3'

TNC-v-AD1 Upper primer 5' – TCA CTG CAT GAG TCC CAG CAA TTC - 3' 74

Lower primer 5' – TTC TCC ACC AAG CCT GTG ATG TGA - 3'

TNC-v-C Upper primer 5' – GGA CCC CCA GGA ATT CAC ACT TT - 3' 72

Lower primer 5' – CCA ATG CCA GTT ATG AGG CCT CTA A - 3'

TNC-v-D Upper primer 5' – TGA GCC ACT GGA AAT AAC CCT ACT T - 3' 111

Lower primer 5' – CGC CTT CCT TTG CTT ATT CCA TA - 3'

TBP Upper primer 5' – TGC ACA GGA GCC AAG AGT GAA – 3' 132 Lower primer 5' – CAC ATC ACA GCT CCC CAC CA– 3' RPLP0 Upper primer 5’ – GGC GAC CTG GAA GTC CAA CT – 3’ 149

Lower primer 5' – CCA TCA GCA CCA CAG CCT TC – 3'

Supplementary table S3 : Genes differentially expressed between plexifom neurofibromas and MPNSTs (P<10-7)

Sequence Name Sequence Code Sequence Description Ratio

ANAPC11 Q9NYG5 Anaphase promoting complex subunit 11 homolog (yeast), an anaphase-promoting complex component that + contains a RING finger domain, displays ubiquitin activity, interacts with the cullin domain of APC2, expression is upregulated in some cancers AP2B1 P21851 Adaptor-related protein complex 2 beta 1 subunit, a large subunit of the AP2 adaptor complex, involved + in clathrin coat assembly at the plasma membrane; may bind to the transmembrane envelope protein of HIV-1 and HTLV retroviruses BIRC5 O15392 Survivin, a member of the inhibitor of apoptosis that is involved in G2-M transition and + exit of the mitotic cell cycle; may play a role in oncogenesis BOP1 AAH13980.1 Block of proliferation 1, has four WD40 repeats and PEST sequences, which likely mediate protein- + protein interactions, has strong similarity to mouse Bop1, which is a protein involved in 28S and 5.8S rRNA processing and large ribosomal subunit biogenesis CBX7 O95931 Protein containing a Chromo domain, which bind chromatin, has moderate similarity to a region of - chromobox homolog 4 Drosophila Pc class (human CBX4), which is a component of the HPC-HPH Polycomb group complex and functions as a transcriptional repressor CCNB2 O95067 Cyclin B2, a CDC2 kinase-associated cyclin that is involved in Golgi apparatus disassembly, may function + in p53 (TP53) -mediated cell cycle arrest at the G2/M transition, may mediate cell cycle arrest by linking CDC2 with TGFbeta type II receptor (TGFBR2) CCND2 P30279 Cyclin D2, a member of the cyclin family of CDK regulatory subunits, forms active kinase complexes with + cyclin dependent kinases that phosphorylate RB1 to promote G1 to S-phase progression, plays a role in viral infection and oncogenesis CDK4 AAH15669.1 Cyclin dependent kinase 4, complex with cyclin D1(CCND1)is required for cell cycle progression, + phosphorylates Rb1 and inactivates its repressor function; mutation in corresponding gene is associated with familial melanoma tumors CKS2 P33552 Protein that binds to CDC2/CDC28 protein kinase, regulates CDK-cyclin complexes; similar to S. pombe + p13suc1 CSNK2B P13862 Casein kinase II beta subunit, regulatory subunit of casein kinase II (CK2), confers stability and + specificity to CK2 catalytic subunits, may mediate formation of the tetrameric CK2 complex, and may be involved in heat stress response DC6 AAF89829.1 Protein of unknown function + DF AAA35527.1 Adipsin (complement factor D), a serine protease that functions in the alternative complement pathway - to cleave complement factor B when complexed to component C3, deficiency in humans results in increased susceptibility to Neisseria meningitidis DIA1 P00387 NADH-dependent cytochrome b5 reductase (diaphorase), soluble erythrocyte-specific form functions in - methemoglobin reduction, ubiquitous membrane-bound form functions in lipid ; mutations in the gene cause methemoglobinemia types I and II DNMT3A Q9Y6K1 DNA methyltransferase 3A, a putative DNA (cytosine-5-)-methyltransferase; corresponding gene is + overexpressed in leukemias DPT Q07507 Dermatopontin, a putative component of the extracellular matrix that is involved in cell adhesion, - regulated by various cytokines and matrix collagen, may be associated with systemic sclerosis and hypertrophic scar fibrosis DTYMK P23919 Deoxythymidylate kinase (dTMP kinase), phosphorylates dTMP to dTDP in dTTP biosynthesis, activity + and transcript abundance peak in S phase, rate limiting for activating zidovudine (AZT) EDG1 P21453 Endothelial differentiation gene-1, a G protein-coupled receptor activated by sphingosine-1-phosphate, - signals through Gi proteins and Rac, Ras and Rho signaling pathways, promotes cell adhesion, migration, and angiogenesis, inhibits apoptosis EPAS1 Q99814 Endothelial PAS domain protein 1, a transcription factor that forms a heterodimer with aryl - hydrocarbon receptor nuclear translocator (ARNT) to activate hypoxia inducible promoters, may play a role in angiogenesis during embryogenesis and tumorigenesis FLJ10044 BAA91419.1 Protein containing LIM domains, which may mediate protein-protein interactions - FLJ14050 BAB14829.1 Protein of unknown function - FLJ20401 AAH21860.1 Protein containing a DIL domain, which are found in dilute myosin heavy chain proteins - FLJ20535 BAA91242.1 Protein containing three TPR (tetratrico peptide repeat) domains, which may mediate protein-protein - interactions FLJ20539 BAA91245.1 Protein of unknown function, has low similarity to uncharacterized human KIAA1906 + FLJ22626 BAB15428.1 Protein of unknown function, has low similarity to uncharacterized C. elegans E01A2.4 + FOXO1A Q12778 Forkhead box O1A, a transcriptional activator as well as a transcriptional co-activator and corepressor; - a translocation resulting in fusion to PAX3 or PAX7 is associated with alveolar rhabdomyosarcoma FY Q16570 Duffy blood group, a C-C and C-X-C chemokine receptor for interleukin 8 (IL8) and malarial Plasmodium - parasites, may play a role in the interactions between granulocytes and postcapillary epithelial cells GPR56 AAH08770.1 G protein-coupled receptor 56, a putative G protein-coupled receptor that may function in cell adhesion, - cell-cell signaling, and is differentially expressed during metastatic progression of melanomas H2BFD P06899 H2B histone family member D, a component of nucleosomes, along with core histones H2A, H3, H4 and + DNA HMG4 O15347 High-mobility group (nonhistone chromosomal) protein 4, member of the HMG 1/2 family, which have a + common DNA-binding motif and bind DNA with low specificity and facilitate assembly of nucleoprotein complexes on bent DNA, may play a role in development HMGIC P52926 High mobility group I protein C, a chromatin component with AT-hook DNA-binding motifs, may play a + role in cell proliferation and adipogenesis; variants are associated with benign mesenchymal tumors and sarcomas HSPB2 Q16082 Heat shock protein 2 (27kD), a member of the alpha-crystallin/small heat shock protein family, - interacts with myotonic dystrophy protein kinase (DMPK) and protects it from heat shock; may play a role in myotonic dystrophy and chemoresistance I_1000035 AAH26078.1 Protein with high similarity to human C2orf6, which forms stable complexes with striatin (STRN)- - protein phosphatase 2A and GS2NA-protein phosphatase 2A and may participate in cytoskeletal regulation and cytokinesis, member of the Mob1 and phocein family I_1000690 AAB34266.1 Protein with high similarity to mouse Tcf15, which is a transcription factor required for regulation of + somite morphogenesis and subsequent patterning of the axial skeleton and skeletal muscles, contains a helix-loop-helix DNA-binding domain I_957430 CAC39772.1 Member of the SCP-like extracellular protein family, has a region of moderate similarity to late - gestation lung protein (rat Rn.4346), which may regulate lung development by acting as a protease inhibitor and regulating extracellular matrix degradation I_960223 AAH00322.1 Protein of unknown function, has very strong similarity to uncharacterized human DEEPEST + I_964086 AK022617.1 Protein of unknown function + I_965586 I_965586 Protein of unknown function - I_966202 AAH06551.1 Protein with high similarity to lamin B1 (human LMNB1), which may be a structural member of the + nuclear lamina cleaved by proteases during apoptosis, member of the intermediate filament family, contains an intermediate filament tail domain IGF1 P01343 Insulin-like growth factor 1, ligand for the protein tyrosine kinase-linked IGF receptor (IGF1R), - stimulates cell proliferation and inhibits apoptosis, plays a role in embyrogenesis and morphogenesis; may play a role in neoplastic transformation ITGB4 CAA36433.1 Integrin beta 4 subunit, binds integrin alpha 6 (ITGA6) to form a receptor that anchors epithelial cells - to the basal lamina, loss of function contributes to metastasis of carcinomas, deficiency is associated with epidermolysis bullosa and pyloric atresia KDELR2 P33947 KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention receptor 2, retrieves proteins from + the Golgi apparatus for retrograde transport to the endoplasmic reticulum, overexpression disrupts vesicular trafficking KPNB1 Q14974 Importin beta (karyopherin beta 1), a subunit of the NLS (nuclear localization signal) receptor complex, + binds to the nuclear pore complex and mediates translocation of the importin alpha-NLS complex into the nucleus LOC51159 AAF00091.1 Protein of unknown function - MAGED1 Q9Y5V3 Melanoma antigen family D 1, a novel member of the melanoma antigen family D that is expressed in + many normal tissues, unlike other MAGE family members MCM3 P25205 Minichromosome maintenance deficient 3, associates with the DNA polymerase alpha-primase complex, + involved in the initiation of DNA replication, selectively degraded during apoptosis MCM6 Q14566 Minichromosome maintenance deficient 6, a member of the MCM family of chromatin-binding proteins, + has ATP-dependent helicase activity, and may have a role in initiation of DNA replication MFAP2 P55001 Microfibrillar-associated protein 2, a component of the elastin-associated microfibrils of the + extracellular matrix; terminal galactose and galactosamine modifications are associated with the metastatic potential of melanoma cells MGC16386 AAH11000.1 Protein of unknown function + OSBPL5 Q9H0X9 Member of the oxysterol-binding protein (OSBP) family, contains a pleckstrin homology (PH) domain, has - a region of low similarity to S. cerevisiae Kes1p, which is implicated in ergosterol biosynthesis OSF-2 BAA02837.1 Osteoblast specific factor 2, a putative cell adhesion molecule that may play a role in homophilic cell + adhesion during bone formation, altered expression may contribute to the development of melorheostosis, a rare bone disease PKMYT1 AAC04478.1 Membrane-associated tyrosine- and threonine-specific CDC2-inhibitory kinase, controls the cell cycle by + phosphorylating and inactivating cyclin-bound CDC2, may play a role in shuttling CDC2-cyclin B1 (CCNB1) complexes between the nucleus and cytoplasm PRAME P78395 Preferentially expressed antigen of melanoma, a tumor antigen which contains epitopes recognized by + histocompatibility leukocyte antigen of cytotoxic T cells, overexpressed in leukemias PRC1 AAH03138.1 Protein regulator of cytokinesis 1, associates with the mitotic spindle and is required for cytokinesis but + not nuclear division, may function in spindle elongation, a substrate for phosphorylation by many cyclin dependent kinases PRO1779 AAA82935.1 Protein with very strong similarity to centromere protein F (mitosin, human CENPF), which is a + centromere-associated protein involved in kinetochore assembly and in chromosome movement during mitosis PTPN9 P43378 Protein tyrosine phosphatase non-receptor type 9, activated by phosphatidylinositols binding, modulated - by neutrophil phagocytosis, may play a role in regulation of phagocytosis and secretory vesicle formation; gene deletion may be associated with autism RRM2 P31350 Ribonucleotide reductase subunit M2, associates with RRM1 to yield an that reduces + ribonucleotides to deoxyribonucleotides, a rate limiting enzyme for DNA synthesis; overexpression is associated with hydroxyurea resistance SCYA14 CAA94309.1 Small inducible cytokine subfamily A member 14, a chemoattractant that enhances proliferation of - myeloid progenitor cells and may affect replication of the HIV 1 virus, may play a role in AIDS pathogenesis and chemokine receptor CCR1 associated diseases SEC61G P38384 Member of the SecE-Sec61-gamma subunits of protein translocation complex family, has very strong + similarity to uncharacterized mouse Sec61g SEMA3A Q14563 Semaphorin-III (collapsin 1/semaphorin D) a chemorepellent involved in axon guidance and likely has a + role in the migration of neurons and glia in the developing brain SH3BP5 O60239 SH3 domain-binding protein which preferentially associates with Btk (Brutons tyrosine kinase), inhibits - BTK activity and B cell receptor-induced, BTK -dependent calcium mobilization, inositol 1,4,5- trisphosphate generation and apoptosis SNRPC P09234 Small nuclear ribonucleoprotein polypeptide C, binds U1 snRNP particle and promotes the formation of + the early spliceosomal complexes, also involved in transcriptional regulation, a frequent target of autoantibodies in systemic lupus erythematosus (SLE) SNX10 Q9Y5X0 Protein containing two phox protein (PX) domains, which bind phosphoinositides + SPP1 P10451 Secreted phosphoprotein-1 (osteopontin), an extracellular matrix glycoprotein and integrin ligand, plays + a role in cellular adhesion and migration, regulates normal and atherosclerotic calcification processes, contributes to tumor invasion and metastasis TCF3 P15923 Transcription factor 3, a helix-loop-helix transcription factor that regulates immunoglobulin gene + expression; chromosomal rearrangements leading to the expression of a E2A - PBX1 chimeric protein are associated with acute leukemias TIMELESS BAA36499.1 Timeless homolog (Drosophila), a protein involved in circadian oscillation autoregulation + TNA P05452 Tetranectin, a member of the C type lectin family and a homotrimeric plasminogen binding protein found - in plasma and bone, may contribute to bone mineralization and skeletal development; serves as a tumor marker, contributing to ovarian cancer prognosis TNC P24821 Tenascin C (hexabrachion), a multidomain extracellular matrix glycoprotein, may be involved in cell + adhesion and neurite outgrowth, induced by hypoxia and has increased expression in colorectal carcinoma, osteosarcoma metastases and pulmonary hypertension TNXB P22105 Tenascin-X, an extracellular matrix protein with EGF-like and fibronectin type III-like repeats; - deficiency is associated with Ehlers-Danlos syndrome, which is a connective tissue disorder TOP2A P11388 DNA topoisomerase II alpha, has diverse roles in DNA replication, chromosome condensation, cell cycle + control; mutations in the corresponding gene confer resistance to topoisomerase II-targeting antitumor drugs TPSB2 P20231 Protein with very strong similarity to tryptase beta 1 (human TPSB1), which is a mast cell serine - protease that activates mast cells and may play a role in asthma pathogenesis, member of the trypsin family of serine proteases TST Q16762 Thiosulfate sulfotransferase (), a thiosulfate sulfotransferase enzyme, functions in - detoxification of cyanide TUBB2 P05218 Beta-2 tubulin, a member of the tubulin family of structural proteins that polymerizes to form + microtubules, soluble form binds nuclear envelope TYMS P04818 Thymidylate synthetase, catalyzes the reductive methylation of dUMP to dTMP with concomitant + conversion of 5,10-methylenetetrahydrofolate to dihydrofolate UCHL1 CAA28443.1 Ubiquitin carboxyl-terminal esterase (ubiquitin thioesterase), a cysteine (thiol) protease, involved in + deubiquitylation; variant forms may be associated with Parkinsons disease in isolated cases, increased expression may be a marker for lung cancer

+ : Gene upregulated in the MPNSTs compared to the plexiform neurofibromas - : Gene downregulated in the MPNSTs compared to the plexiform neurofibromas

Supplementary table S4: mRNA levels of selected genes in dermal neurofibromas, plexiform neurofibromas, and MPNSTs.

Dermal Plexiform MPNSTs a GENES Gene definition neurofibromas neurofibromas P (n=10) (n=20) (n=14)

Up-regulated genes

b BIRC5/Survivin Survivin 1.0 [0.08 - 3.6] 3.6 [0.24 - 19.2] 40.1 [1.3 - 1299] 0.00004 SPP1 Secreted phosphoprotein 1 (osteopontin) 1.0 [0.26 - 71] 2.8 [0.07 - 261] 665 [235 - 2256] 0.00001 TOP2A Topoisomerase II alpha 1.0 [0.21 - 2.9] 3.1 [0.37 – 33.9] 39.0 [1.5 - 406] 0.00002 OSF-2 Osteoblast specific factor 2 (periostin) 1.0 [0.12 - 5.9] 6.3 [0.12 - 82.6] 56.1 [3.2 - 215] 0.0002

Down-regulated genes

ITGB4 Integrin beta 4 1.0 [0.46 - 1.9] 0.72 [0.18 - 3.4] 0.02 [0.00 – 1.3] 0.0004 TPSB Tryptase beta 1.0 [0.09 - 6.6] 1.4 [0.04 - 3.9] 0.06 [0.02 - 0.11] 0.00007

aMann and Whitney U test: dermal neurofibromas vs plexiform neurofibromas vs MPNSTs bMedian [range] of gene mRNA levels