Supplementary Table 1 Symbols and full names of 120 cytokines included in the protein antibody array.
Protein Name / Official gene name provided by HUGO Gene Nomenclature Committee (HGNC)Adiponectin / adiponectin, C1Q and collagen domain containing
AGRP / agouti related protein homolog (mouse)
Amphiregulin / amphiregulin (schwannoma-derived growth factor)
ANG-2 / angiopoietin 2
Angiogenin / angiogenin, ribonuclease, RNase A family, 5
AXL / AXL receptor tyrosine kinase
basic FGF / fibroblast growth factor 2 (basic)
BDNF / brain-derived neurotrophic factor
BMP-4 / bone morphogenetic protein 4
BMP-6 / bone morphogenetic protein 6
BTC / betacellulin
CCL1/I-309 / chemokine (C-C motif) ligand 1
CCL2/MCP-1 / chemokine (C-C motif) ligand 2
CCL3/MIP-1α / chemokine (C-C motif) ligand 3
CCL4/MIP-1β / chemokine (C-C motif) ligand 4
CCL5/RANTES / chemokine (C-C motif) ligand 5
CCL7/MCP-3 / chemokine (C-C motif) ligand 7
CCL8/MCP-2 / chemokine (C-C motif) ligand 8
CCL11/Eotaxin / chemokine (C-C motif) ligand 11
CCL13/MCP-4 / chemokine (C-C motif) ligand 13
CCL15/MIP-1δ / chemokine (C-C motif) ligand 15
CCL16/HCC-4 / chemokine (C-C motif) ligand 16
CCL17/TARC / chemokine (C-C motif) ligand 17
CCL18/PARC / chemokine (C-C motif) ligand 18 (pulmonary and activation-regulated)
CCL19/MIP-3β / chemokine (C-C motif) ligand 19
CCL20/MIP-3α / chemokine (C-C motif) ligand 20
CCL22/MDC / chemokine (C-C motif) ligand 22
CCL23/CKb8-1 / chemokine (C-C motif) ligand 23
CCL24/Eotaxin-2 / chemokine (C-C motif) ligand 24
CCL25/TECK / chemokine (C-C motif) ligand 25
CCL26/Eotaxin-3 / chemokine (C-C motif) ligand 26
CCL27/CTACK / chemokine (C-C motif) ligand 27
CNTF / ciliary neurotrophic factor
CX3CL1/Fractalkine / chemokine (C-X3-C motif) ligand 1
CXCL1,2,3/GRO-α,β,γ / chemokine (C-X-C motif) ligand 1, chemokine (C-X-C motif) ligand 2, chemokine (C-X-C motif) ligand 3
CXCL1/GRO-α / chemokine (C-X-C motif) ligand 1
CXCL5/ENA-78 / chemokine (C-X-C motif) ligand 5
CXCL6/GCP-2 / chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2)
CXCL7/NAP-2 / pro-platelet basic protein (chemokine (C-X-C motif) ligand 7)
CXCL8/IL-8 / interleukin 8
CXCL9/MIG / chemokine (C-X-C motif) ligand 9
CXCL11/I-TAC / chemokine (C-X-C motif) ligand 11
CXCL12/SDF-1 / chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)
CXCL13/BLC / chemokine (C-X-C motif) ligand 13 (B-cell chemoattractant)
EGF / epidermal growth factor (beta-urogastrone)
EGFR / epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)
Fas / Fas (TNF receptor superfamily, member 6)
FGF-4 / fibroblast growth factor 4 (heparin secretory transforming protein 1)
FGF-6 / fibroblast growth factor 6
FGF-7 / fibroblast growth factor 7 (keratinocyte growth factor)
FGF-9 / fibroblast growth factor 9 (glia-activating factor)
Fit-3L / fms-related tyrosine kinase 3 ligand
G-CSF / colony stimulating factor 3 (granulocyte)
GDNF / glial cell derived neurotrophic factor
GITR / tumor necrosis factor receptor superfamily, member 18
GITR-L / tumor necrosis factor (ligand) superfamily, member 18
GM-CSF / colony stimulating factor 2 (granulocyte-macrophage)
HGF / hepatocyte growth factor (hepapoietin A; scatter factor)
ICAM-1 / intercellular adhesion molecule 1 (CD54), human rhinovirus receptor
ICAM-3 / intercellular adhesion molecule 3
IFN-γ / interferon, gamma
IGF1-R / insulin-like growth factor 1 receptor
IGFBP-1 / insulin-like growth factor binding protein 1
IGFBP-2 / insulin-like growth factor binding protein 2
IGFBP-3 / insulin-like growth factor binding protein 3
IGFBP-4 / insulin-like growth factor binding protein 4
IGFBP-6 / insulin-like growth factor binding protein 6
IGF-I / insulin-like growth factor 1 (somatomedin C)
IL-1 R-like 1 / interleukin 1 receptor-like 1, suppression of tumorigenicity 2
IL-1 sRI / interleukin 1 receptor, type I
IL-1ra / interleukin 1 receptor antagonist
IL-1α / interleukin 1, alpha
IL-1β / interleukin 1, beta
IL-2 / interleukin 2
IL-2 sRa / interleukin 2 receptor, alpha
IL-3 / interleukin 3 (colony-stimulating factor, multiple)
IL-4 / interleukin 4
IL-5 / interleukin 5 (colony-stimulating factor, eosinophil)
IL-6 / interleukin 6 (interferon, beta 2)
IL-6 sR / interleukin 6 receptor
IL-7 / interleukin 7
IL-10 / interleukin 10
IL-11 / interleukin 11
IL-12p40 / interleukin 12B (natural killer cell stimulatory factor 2, p40)
IL-12p70 / interleukin 12A (natural killer cell stimulatory factor 1, p35)
IL-13 / interleukin 13
IL-15 / interleukin 15
IL-16 / interleukin 16 (lymphocyte chemoattractant factor)
IL-17 / interleukin 17
Leptin / leptin
LIGHT / tumor necrosis factor (ligand) superfamily, member 14
M-CSF / colony stimulating factor 1 (macrophage)
MIF / macrophage migration inhibitory factor (glycosylation-inhibiting factor)
MSPα-chain / macrophage stimulating 1 (hepatocyte growth factor-like) alpha-chain
NGF-β / nerve growth factor, beta polypeptide
NT-3 / neurotrophin 3
NT-4/5 / neurotrophin 5 (neurotrophin 4/5)
Oncostatin M / oncostatin M
Osteoprotegerin / tumor necrosis factor receptor superfamily, member 11b
PDGF-BB / platelet-derived growth factor beta polypeptide (simian sarcoma viral (v-sis) oncogene homolog)
PLGF / placental growth factor, vascular endothelial growth factor-related protein
SCF / KIT ligand
Sgp130 / interleukin 6 signal transducer (gp130, oncostatin M receptor)
TGF-β1 / transforming growth factor, beta 1
TGF-β3 / transforming growth factor, beta 3
TIMP-1 / Tissue inhibitor of metallopeptidase 1
TIMP-2 / Tissue inhibitor of metallopeptidase 2
TNFR-1 / tumor necrosis factor receptor superfamily, member 1A
TNFR-2 / tumor necrosis factor receptor superfamily, member 14
TNF-α / tumor necrosis factor (TNF superfamily, member 2)
TNF-β / lymphotoxin alpha (TNF superfamily, member 1)
Normalization of cytokine antibody array data:
The data of the cytokine antibody array were first transformed by subtracting local background intensities from each spot, and then the average of the duplicate spots for each protein was normalized to the average of six positive controls on each membrane. The expression data from the two filters per sample were then normalized to the median expression of all 120 proteins followed by Z score transformation. Z scores are calculated by subtracting the mean intensity of the 120 proteins from the raw intensity data for each protein, and dividing that result by the SD of all of the 120 intensity readings.
Transforming signal intensity into concentration:
To validate the prognostic value of these four cytokines, we first quantified the selected cytokines levels in samples of the training set from the densitometry data in cytokine array into actual concentration (pg/ml) using ELISA method. Then the weighting factors (B) were determined (Supplementary table 2). We then condensed the cytokine expression values for each sample into a single value, named cytokine score. Each variable was weighted according to its individual strength of association of survival. The Cox proportional hazards coefficients (B) from Cox regression analysis were used as the weighting factor to calculate the cytokine score according to the formula, where B is the Cox regression coefficient and V is the variable (cytokine expression value). For consistency of comparison, the identical weighting factors and cutoff scores as in the training set were used in all subgroups (LGG, AA, GBM) involved in the subsequent validation analysis.
Supplementary table 2. Concentration of four cytokines in the training set and their correlation with patients’ survival.
Cytokine / Concentration (pg/ml, mean±SD) / P value / B / Exp(B) (95%CI)IL-7 / 62.39±28.15 / 0.001 / -0.032 / 0.968 (0.950-0.987)
IL1R4/ST2 / 60.33±18.60 / 0.008 / 0.029 / 1.029 (1.008-1.051)
MCP-1 / 44.42±23.26 / 0.263 / 0.009 / 1.009 (0.993-1.026)
sgp130 / 210.00±24.12 / 0.058 / -0.020 / 0.981 (0.961-1.001)
Supplementary Table3 Multivariate Cox regression analysis for predictors of overall survival in the 45 GBM patients of the training set.
Variable / Value (mean±SD) / Hazard ratio (95% CI) / pAge
Gender (male/female) / 43.87±13.81
33/12 / 1.008 (0.974, 1.043)
0.696 (0.280-1.730) / 0.640
0.436
Preoperational KPS / 75.65±12.58 / 1.007 (0.967, 1.049) / 0.741
Extent of resection / 1.67±0.61 / 1.114 (0.499, 2.488) / 0.791
4-cytokine score / -4.14±1.45 / 2.068 (1.357, 3.153) / 0.001
In the validation set, plasma concentrations of the four cytokines were assayed with ELISA. Cox-regression analyses adjusting for common clinical predictors (age, preoperational KPS score, extent of resection) were conducted for each of these cytokines. Concentration differences among different grades were also compared (Supplementary table 4).
Supplementary table 4 Correlation of four cytokines as individual markers with patients’ survival and tumor grade in the validation set.
Cytokine / Group / Concentration (pg/ml) / Correlation with survival (Cox-regression analysis) / Correlation with tumor grade (One-way ANOVA)p-value / HR (95%CI) / p-value
IL-7 / Glioma / 64.46±35.11 / 0.009 / 0.984 (0.973-0.996)
Grade II / 56.21±32.58 / 0.341 / 0.982 (0.946-1.019) / 0.071
Grade III / 66.06±39.14 / 0.076 / 0.950 (0.899-1.005)
Grade IV / 69.32±32.92 / 0.009 / 0.980 (0.965-0.995)
IL1R4 / Glioma / 53.22±156.87 / 0.005 / 1.001 (1.000-1.002)
Grade II / 34.32±63.00 / 0.183 / 1.008 (0.996-1.020) / 0.392
Grade III / 50.58±128.02 / 0.852 / 1.000 (0.996-1.004)
Grade IV / 68.16±212.01 / 0.007 / 1.002 (1.000-1.003)
MCP-1 / Glioma / 40.78±27.25 / 0.724 / 1.002 (0.992-1.012)
Grade II / 44.07±35.76 / 0.607 / 0.995 (0..978-1.013) / 0.378
Grade III / 41.24±21.76 / 0.374 / 0.986 (0.954-1.018)
Grade IV / 38.14±23.84 / 0.018 / 1.020 (1.003-1.036)
sgp130 / Glioma / 211.12±45.16 / 0.078 / 0.994 (0.988-1.001)
Grade II / 198.50±41.65 / 0.206 / 0.988 (0.970-1.007) / 0.004
Grade III / 210.06±46.11 / 0.067 / 0.985 (0.969-1.001)
Grade IV / 221.26±44.88 / 0.373 / 0.996 (0.987-1.005)