Figure S1. Two-variable Prediction model for high-risk IPMN – ROC curve.
Red dot marks the sensitivity and specificity of the radiographic MD-/BD- IPMN subtype alone.
Table S1. Complete list of multiplexed biomarker assays (87 markers).
Biological groups Markers
Tumor markers Alpha-fetoprotein (AFP), CA 19–9, CA 125,
CA 15–3, CA72–4, CEA
Hormones Adrenocorticotropic hormone (ACTH), follicle
stimulating hormone (FSH),
growth hormone (GH), insulin, luteinizing hormone (LH), prolactin (PRL),
parathyroid hormone (PTH), thyroid stimulating hormone (TSH), pancreatic polypeptide (PP), peptide YY (PYY, total) Apoptotic factors Cytokeratin 19 (Cyfra 21–1), DR5, sFas, sFasL
Cell Adhesion ICAM-1, VCAM-1, NCAM
Proteases/inhibitors Kallikrein 10, MMP-2, 3, 9, TIMP-1–4, Cathepsin D
Cytokines/chemokines/
receptors
CD40L, eotaxin-1, fractalkine, IFNγ, IL-10, IL-12p70, IL-13, IL-1β, IL-1Ra,
IL-2, IL-2R, sIL-2Rα, IL-4, IL-5, IL-6, IL-6R, IL-7, IL-8, IP-10, macrophage inhibitory factor (MIF),
MIP-1b, TGFα, TNFα, TNF-RI, TNF-RII Growth/angiogenesis
factors/receptors
Angiostatin, EGFR, endostatin, ErbB2, GM- CSF, IGFBP-1, thrombospondin, CNTF, NGF, BDNF
Adipokines Adiponectin, leptin
Apolipoproteins ApoAI, ApoAII, ApoB, ApoCII, ApoCIII,
ApoE
Other CRP, ghrelin (active), gastric inhibitory
polypeptide (GIP, total), glucagon like peptide 1(GLP-1, active), HE4, mesothelin, osteocalcin (OC), osteoprotegerin (OPG), osteopontin (OPN),
serum amyloid A (SAA), serum amyloid P (SAP), transglutaminase II (TG II), total plasminogen
activator inhibitor 1 (tPAI 1), myeloperoxidase (MPO)
Table S2. Cyst fluid proteins stratified by low-risk and high-risk groups.
Cyst fluid proteins n=78 Low-risk (n=48) High-risk (n=30) p-value
MIF, pg/ml 77 8163.6 (47) 17164 (30) 0.003
FASL, pg/ml 77 39 (47) 56.3 (30) 0.1
Fas, pg/ml 77 1947.6 (47) 6018.5 (30) 0.4
VCAM-1, pg/ml 78 12397.6 (48) 17091.2 (30) 0.1
ICAM-1, pg/ml 78 10140.2 (48) 12075.8 (30) 0.4
MMP-9, pg/ml 78 78728.1 (48) 156359.2 (30) 0.03
MPO, pg/ml 78 756696.40 (48) 1429481.3 (30) 0.009
tPAI-1, pg/ml 78 6696 (48) 8430.3 (30) 0.2
MIP-4, pg/ml 78 8466.8 (48) 9366.1 (30) 0.1
CathepsinD, pg/ml 78 401810.1 (48) 809082.5 (30) 0.01
NCAM, pg/ml 78 3222 (48) 9840.2 (30) 0.05
OPG, pg/ml 76 5840.3 (47) 12224.1 (29) 0.5
OC, pg/ml 76 3919.9 (47) 11140.2 (29) 0.01
OPN, pg/ml 76 10726.7 (47) 11399.8 (29) 0.9
PTH, pg/ml 76 109.9 (47) 326.2 (29) 0.001
Clusterin, pg/ml 78 9288731.2 (48) 13572936.1 (30) 0.5
M-CSF, pg/ml 76 58386.3 (48) 8962.6 (28) 0.5
Fractalkine, pg/ml 74 270.9 (46) 709.7 (28) 0.1
IFNα, pg/ml 74 40.9 (46) 86.7 (28) 0.1
IFNγ, pg/ml 74 6.8 (46) 27.5 (28) 0.003
IL-1α, pg/ml 74 79.1 (46) 694.7 (28) 0.02
IL-1β, pg/ml 74 92.9 (46) 707.1 (28) 0.001
IL-1Ra, pg/ml 74 482.8 (46) 1012.3 (28) 0.03
IL-2, pg/ml 74 2.8 (46) 15.2 (28) 0.001
IL-4, pg/ml 74 3.8 (46) 31 (28) 0.004
IL-5, pg/ml 74 0.5 (46) 6.8 (28) 0.001
IL-6, pg/ml 74 642.8 (46) 714.5 (28) 0.3
IL-7, pg/ml 74 14.9 (46) 31 (28) 0.1
CD40L, pg/ml 74 375.6 (46) 979.3 (28) 0.005
sIL-2Rα, pg/ml 74 13.1 (46) 86.5 (28) 0.003
TGFα, pg/ml 74 18.8 (46) 25.1 (28) 0.5
TNFα, pg/ml 74 24.6 (46) 117.6 (28) 0.1
TNFβ, pg/ml 74 2 (46) 20.5 (28) 0.013
Adiponectin, pg/ml 71 198940.5 (43) 476827.8 (28) 0.3
Lipocalin/NGAL, pg/ml 71 1610013 (43) 1469529.8 (28) 0.5
NGF, pg/ml 63 9.3 (39) 46.5 (24) 0.049
HGF, pg/ml 63 6756.1 (39) 4050 (24) 0.5
ACTH, pg/ml 71 9.6 (43) 17.3 (28) 0.2
CNTF, pg/ml 71 440.2 (43) 1269.5 (28) 0.03
FSH, uIU/ml 71 2657.4 (43) 4786.5 (28) 0.3
GH, pg/ml 71 72.5 (43) 209 (28) 0.08
LH, uIU/ml 71 402.5 (43) 1388.6 (28) 0.2
TSH, nIU/ml 71 469 (43) 5966.8 (28) 0.02
Prolactin, pg/ml 71 1037.5 (43) 8035.9 (28) 0.4
BDNF, pg/ml 71 107.7 (43) 335.2 (28) 0.05
Apolipoprotein Apo AII , pg/ml
78 2903.6 (48) 4942.9 (30) 0.5
Apolipoprotein Apo B, pg/ml
78 13165.4 (48) 15593.3 (30) 0.3
Apolipoprotein A1, pg/ml 78 113907.9 (48) 276089.3 (30) 0.3 Apolipoprotein Apo CIII,
pg/ml 78 675.9 (48) 2484.2 (30) 0.04
Apolipoprotein Apo E,
pg/ml 78 448.5 (48) 1665 (30) 0.5
CRP, pg/ml 78 294595 (48) 393225.2 (30) 0.1
SAA, pg/ml 78 98033.9 (48) 149759.9 (30) 0.1
SAP, pg/ml 78 783565.9 (48) 2221856.9 (30) 0.03
SCC, pg/ml 73 155586.7 (45) 1075347.2 (28) 0.1
TTR, pg/ml 73 4685380.5 (45) 7257130.9 (28) 0.5
CA72-4, U/ml 73 62.1 (45) 399.2 (28) 0.07
AFP, pg/ml 73 218.2 (45) 4837.9 (28) 0.009
Kallikrein, pg/ml 73 39373.8 (45) 42204.1 (28) 0.2
Mesothelin, pg/ml 73 92544.8 (45) 117811.9 (28) 0.5
IGFBP-1, pg/ml 73 198519.8 (45) 129304.8 (28) 0.8
Cytokeratin 19, pg/ml 73 191305.7 (45) 302619.5 (28) 0.02
CA-125, U/mL 73 486.8 (45) 371.3 (28) 0.7
CEA, pg/ml 73 382594.7 (45) 322379.4 (28) 0.2
ErbB2, pg/ml 73 4420.8 (45) 11458.6 (28) 0.01
EGFR, pg/ml 73 3430.8 (45) 15157.7 (28) 0.003
CA19-9, U/ml 73 372.5 (45) 234.5 (28) 0.11
MMP-2, pg/ml 75 25094.8 (47) 14786.3 (28) 0.9
MMP-3, pg/ml 75 2185.8 (47) 8824.3 (28) 0.2
TIMP-1, pg/ml 74 197682.3 (46) 242740.8 (28) 0.9
TIMP-2, pg/ml 74 38141.9 (46) 33539.5 (28) 0.5
TIMP-3, pg/ml 74 8070.5 (46) 9615.2 (28) 0.2
TIMP-4, pg/ml 74 358.8 (46) 619.3 (28) 0.8
TNF-R2, pg/ml 56 414.8 (34) 726.7 (22) 0.08
TNF-R1, pg/ml 56 715.8 (34) 1305.4 (22) 0.3
Eotaxin, pg/ml 56 18.6 (34) 33.9 (22) 0.8
MIP-1B, pg/ml 56 67.1 (34) 187.8 (22) 0.07
TNFα, pg/ml 56 7.5 (34) 35.6 (22) 0.02
IP-10, pg/ml 56 163.2 (34) 300.7 (22) 0.1
IL-2R, pg/ml 56 71.8 (34) 261.7 (22) 0.01
IL-8, pg/ml 56 10178.5 (34) 18625.5 (22) 0.4
Tg II, pg/ml 65 2372.3 (39) 4341.2 (26) 0.1
HE4, pg/ml 50 177274.9 (31) 149787.5 (19) 0.7
CA15-3, pg/ml 50 69.2 (31) 9.5 (19) 0.9
Thrombospondin, pg/ml 62 332915 (38) 397526 (24) 0.3
Endostatin, pg/ml 62 24188.2 (38) 30073 (24) 0.2
Angiostatin, pg/ml 62 118222.8 (38) 570947.6 (24) 0.3
Continous variables presented as: mean (number of patients).
Analysis of cyst fluid proteins was performed with Mann-Whitney test.
Table S3. Cyst fluid proteins stratified by Tumor-associated neutrophils (TAN)levels.
# Cyst fluid proteins2
TAN neg (n=52) TAN low (n=9) TAN high (n=16) p-
value1
1 IL-1β, pg/ml 1; 1-2 (50) 1; 1-235 (9) 19.5; 2-950 (14) 0.005
2 IL-1α, pg/ml 1; 1-9 (50) 1; 1-271 (9) 17; 1-760 (14) 0.03
3 IL-2, pg/ml 1; 1-1 (50) 1; 1-6.5 (9) 7.5; 2-26 (14) <0.001
4 sIL-2Rα, pg/ml 0; 0-1 (50) 1; 0-32 (9) 9.5; 1-120 (14) 0.004
5 IL-4, pg/ml 1; 1-2 (50) 4; 1.5-19 (9) 9; 1-35 (14) 0.004
6 IL-5, pg/ml 0; 0-0 (50) 0; 0-1 (9) 1; 0-3.5 (14) 0.004
7 TNFα, pg/ml 2.5; 1-19 (50) 5; 1-167 (9) 20.5; 3-123 (14) 0.02
8 TNFβ, pg/ml 1; 1-1 (50) 1; 1-1.5 (9) 1.5; 1-25 (14) 0.007
9 NGF, pg/ml 0; 0-5 (44) 0; 0-4 (6) 27; 0-113 (13) 0.02
10 NCAM, pg/ml 1500; 1300-2500 (52) 1600; 1400-13000 (9) 2200; 1600-26000 (16) 0.04 11 MPO, pg/ml 97000; 16000-
830000(52) 86000; 17000-
1500000 (9) 2000000; 125000-2700000
(16) 0.04
12 CNTF, pg/ml 177; 150-230 (47) 290; 150-900 (7) 430; 190-3000 (16) 0.02 13 TSH, nIU/ml 31; 26-36 (47) 32; 25-700 (7) 600; 30-25000 (16) 0.02 14 Cathepsin D,
pg/ml 130000; 90000-
430000 (52) 500000; 180000-
900000 (9) 460000; 210000-1200000
(16) 0.02
15 OPG, pg/ml 325; 134-1300 (51) 380; 40-2200 (8) 1700; 600-7200 (16) 0.04 16 OC, pg/ml 75; 50-2300 (51) 1800; 60-8000 (8) 8600; 800-26000 (16) 0.02
17 PTH, pg/ml 7; 4-42 (51) 63; 5-134 (8) 183; 34-685 (16) 0.001
18 IFNγ, pg/ml 2; 1-6 (50) 8; 2.5-13 (9) 10.5; 3-43 (14) 0.005
19 CD40L, pg/ml 16; 3-140 (50) 13; 7-2000 (9) 380; 50-560 (14) 0.04 20 CRP, pg/ml 40000; 18000-160000
(52)
140000; 16000- 500000 (9)
230000; 96000-680000 (16) 0.008 21 EGFR, pg/ml 514; 116-2000 (49) 3000; 200-7000 (7) 2400; 460-12000 (16) 0.02
Numbers presented as: median; interquartile range (number of patients).
1Analysis performed with Kruskal-Walis 1-way ANOVA.
2All proteins are CFIM except: TSH, OC, PTH.
Table S4. Differentially expressed cyst fluid proteins1 stratified by IPMN sub-type.
BD-IPMN (n=43)
# Cyst fluid proteins Functional group Low-risk, (n=35)
High-risk, (n=8)
p-value
1 MIF, pg/ml Immune modulator 1477; 530-550
(33)
4711; 2800- 18000 (7)
0.03
2 CathepsinD, pg/ml Protease 110000; 44000-
340000 (33) 312000;
165000-734000 (7)
0.04
3 CEA, pg/ml Tumor marker 412000; 95000-
630000 (33)
22000; 13000- 73000 (7)
0.01 4 ErbB2, ng/mL Growth factors/cell
cycle
700; 280-2373
(33) 2500; 788-5078
(7) 0.03
5 IL-2R, pg/ml Chemokines/cytokines 41; 41-46 (33) 51; 41-330 (7) 0.03 MD-IPMN (n=35)
# Cyst fluid protein Functional group Low-risk, (n=13)
High-risk, (n=22)
p-value 1 IFNγ, pg/ml Chemokines/cytokines 1.5; 1-2 (12) 10.5; 3-49 (20) <0.001 2 IL-1β, pg/ml Chemokines/cytokines 1; 1-2 (12) 19.5; 1-450 (20) 0.02 3 IL-2, pg/ml Chemokines/cytokines 1; 1-2 (12) 4.5; 1-22.75 (20) 0.01 4 IL-4, pg/ml Chemokines/cytokines 1.5; 1-5 (12) 12.5; 1.25-4.25
(20)
0.01 5 IL-5, pg/ml Chemokines/cytokines 0; 0-0 (12) 1; 0-5.75 (20) 0.009 6 CD-40L, pg/ml Chemokines/cytokines 58.5; 2-325 (12) 380; 43.75-786
(20)
0.04 7 sIL-2Rα, pg/ml Chemokines/cytokines 0.5; 0-1 (12) 9.5; 1-141.75
(20) 0.03
8 TNFβ, pg/ml Chemokines/cytokines 1; 1-1 (12) 1.5; 1-46.25 (20) 0.04 Analysis of cyst fluid proteins was performed with Mann-Whitney test. Presented as: median;
interquartile range (number of patients).
1All proteins are CFIM except: CEA.
Table S5. Multivariate Analysis of risk factors for high-risk IPMN.
Variable OR CI: 95% p-value
Standardized CA72-4
3.5 1.04-12.1 0.04
Standardized MMP9
- - NS
Radiographic
MD-/BD- IPMN 12.4 3.6-41.9 <0.001
Solid
component - - NS
Gender - - NS
Diabetes - - NS