Day +1 Day +7 Day +14




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Day +1

Day +7

Day +14

IL-2Ra & HGF

75th centiles



25%

(95% CI 9-66)



44%

(95% CI 26-74)



60%

(95% CI 41-85)



IL-2Ra & HGF

<75th centiles

39.5%

(95% CI 32-48)



37%

(95% CI 28-48)



28%

(95% CI 20-38)



Supplementary Table 1. Cumulative incidence of GVHD II-IV according to IL-2R and HGF serum concentrations at days +1, +7 and +14








MFD


MUD

P

Number of

samples/time points


TNFR1

Day -1

NA

161

Day +1

917.5

1515.4

<0.001

157

Day +7

908.4

1517

0.06

153

Day +14

1496.5

1945.7

0.28

157

Day +21

1337.3

1753.8

0.01

150

Day +28

1051.9

1728.5

0.05

100

Day +60

694.6

1079.1

0.13

94


IL-2Rα

Day -1

NA

161

Day +1

231.9

603.3

<0.001

157

Day +7

306.7

504.4

0.01

153

Day +14

687.3

564

0.77

157

Day +21

432.2

610.4

0.03

150

Day +28

394.4

529.6

0.01

100

Day +60

240.4

292

0.24

94


HGF

Day -1

NA

161

Day +1

392.5

253.9

<0.001

157

Day +7

343.7

337.8

0.91

153

Day +14

394.3

423.5

0.84

157

Day +21

501

442.2

0.76

150

Day +28

477.3

404.2

0.14

100

Day +60

399.9

429.7

0.14

94


CCL8

Day -1

NA

161

Day +1

10.1

13.4

0.01

157

Day +7

6.5

11.4

0.01

153

Day +14

6.8

16.8

<0.001

157

Day +21

5

9.6

<0.001

150

Day +28

4.5

7.8

<0.001

100

Day +60

7.5

7.8

0.13

94


IL-8

Day -1

NA

161

Day +1

10.6

20.7

<0.001

157

Day +7

16.9

38

<0.001

153

Day +14

20.6

28.5

0.12

157

Day +21

14.4

22.2

0.03

150

Day +28

15.6

17.1

0.17

100

Day +60

12.2

20

0.15

94


IL-12

Day -1

NA

161

Day +1

2.3

2.7

0.63

157

Day +7

1.5

2.3

0.48

153

Day +14

1.9

2.1

0.34

157

Day +21

2.3

2.4

0.44

150

Day +28

2.2

2.4

0.61

100

Day +60

2.8

2.6

0.62

94

Supplementary Table 2. Absolute cytokine serum concentration at each time point. NA: not applicable. The differences between groups was calculated by the Mann-Whitney test.










HSC source

Myeloablative conditioning1







BM

PB

CB

P

Yes

No

P







TNFR1

Day -1

NA

1085

1276

<0.001







Day +1

1081.5

3290

1172.2

<0.001

1116.6

1942.5

<0.001







Day +7

1257.2

2308.9

1237.7

0.05

1237.7

2042.5

0.22







Day +14

1713.4

3232.1

1293.4

<0.001

1765.1

1927.5

0.64







Day +21

1686.3

1974.2

1292.3

0.18

1608.1

1832.6

0.64







Day +28

1418.5

2391.1

1681.9

0.16

1514.9

2032.5

0.86







Day +60

861.7

1641

918.6

0.87

899.5

1176.3

0.32







IL-2Rα

Day -1

NA

283.1

377.7

<0.001







Day +1

386.6

868.3

471.4

<0.001

384.6

695.7

<0.001







Day +7

410.7

825.1

545.3

0.01

421.1

615.1

0.21







Day +14

558.6

907.9

674.5

0.12

559

745.7

0.83







Day +21

541

715.7

623

0.92

566

589.6

0.53







Day +28

428.2

696.1

538

0.18

480.8

513.3

0.82







Day +60

234.9

676.4

252.9

0.96

271.3

237.2

0.34







HGF

Day -1

NA

479.6

315.9

0.01







Day +1

290.3

342.8

266.2

0.09

268

427.6

0.01







Day +7

335.4

486.1

240.4

0.07

312.3

417

0.51







Day +14

423.5

669.2

275.7

<0.001

393.4

435.5

0.35







Day +21

476.4

749.8

330.9

0.13

439.7

502.9

0.93







Day +28

436

826.1

317.7

0.15

436

406.5

0.80







Day +60

412.1

679.3

390.8

0.97

448

299.4

0.11







CCL8

Day -1

NA

11.2

9.3

0.17







Day +1

11.8

11.3

11.6

0.86

12.2

9.6

0.11







Day +7

8.6

7.5

12.2

0.51

8.5

8.9

0.40







Day +14

12.8

14.5

9.4

0.28

13.4

11.2

0.12







Day +21

7.4

8.3

5.5

0.81

7.3

7.9

0.77







Day +28

7.4

7.6

5.2

0.50

6.1

7.8

0.95







Day +60

7.7

8.1

10.5

0.84

7.7

7.7

0.43







IL-8

Day -1

NA

17.1

28.7

<0.001







Day +1

15.2

30.8

15.9

0.13

15.5

19.3

0.19







Day +7

23.5

45

38.3

0.22

27.9

26.9

0.09







Day +14

22

38.3

18.7

0.20

22.5

26.9

0.16







Day +21

16.3

38

16.7

0.79

15.2

22.8

0.42







Day +28

14.4

23

31.2

0.36

15.1

22.4

0.43







Day +60

14.6

22.2

25.1

0.59

17.1

17.1

0.64







IL-12p70

Day -1

NA

2

2.5

<0.001







Day +1

3.1

2.4

1.3

0.54

2.4

3.8

0.02







Day +7

2.1

2

3

0.58

2

2.3

0.16







Day +14

2.1

1.6

3.2

0.74

2

2.4

0.73







Day +21

2.4

2.7

2

0.31

2.3

2.9

0.52







Day +28

2.4

2.5

2.7

0.91

2.4

3.3

0.77







Day +60

2.9

2.9

1.9

0.65

2.6

4.4

0.76






Supplementary Table 3. Cytokine serum concentration according to HSC source or conditioning intensity at each time point. All results are expressed as pg/ml. 1Myeloablative conditioning was considered TBI-based 9.9 Gy, full dose Busulfan-based or Busilvex-based or a combination of drugs containing Thiotepa above 10 mg/Kg. NA: not applicable. The differences between groups were calculated by the Mann-Whitney test.











Patient age

Donor age







9.7 y

<9.7 y

P

21.9 y

<21.9 y

P




TNFR1

Day -1

1276

1045.4

<0.001

NA




Day +1

1453.9

1074.5

0.01

1555.8

946.6

0.01




Day +7

1574.7

1217.1

0.05

1593.6

1063.9

0.05




Day +14

2193.7

1409.7

0.04

2027.9

1241.1

0.04




Day +21

1927

1495.5

0.04

1961.6

1318.6

0.04




Day +28

1921

1536

0.26

1700.4

1241.2

0.28




Day +60

910.7

919.9

0.87

980

764.4

0.87




IL-2Rα

Day -1

263.7

314.1

0.74

NA




Day +1

493.6

430

0.19

536.9

275.5

0.22




Day +7

428

454.7

0.42

500

382.2

0.43




Day +14

598.4

587.6

0.90

559.5

619.1

0.99




Day +21

665.2

537.4

0.35

579.7

523.1

0.28




Day +28

531.5

454.9

0.80

513.3

467.5

0.83




Day +60

212.2

275.1

0.04

317

252.7

0.06




HGF

Day -1

467.8

390.8

0.03

NA










Day +1

320.6

268

0.17

285

309

0.23




Day +7

387.9

289.9

0.07

382.2

283.9

0.07




Day +14

458.7

349.8

0.03

441.3

336

0.02




Day +21

562.7

401.7

0.04

501

402.2

0.06




Day +28

476.8

363.9

0.99

449.9

365.8

0.88




Day +60

448

412.1

0.08

430.1

417.1

0.10




CCL8

Day -1

10.6

10.1

0.17

NA




Day +1

11.2

11.7

0.73

13.2

9.9

0.80




Day +7

9.2

8

0.87

11.1

6.4

0.76




Day +14

14.1

11.8

0.47

16.1

7.2

0.55




Day +21

7.6

6.9

0.32

9.6

5.1

0.34




Day +28

7

7.4

0.65

7.7

5.2

0.71




Day +60

7.2

8.3

0.04

7.7

7.7

0.88




IL-8

Day -1

18.2

20.9

0.55

NA




Day +1

22

14.3

0.09

25.1

10.5

0.13




Day +7

28.3

26.6

0.71

35.4

18.3

0.71




Day +14

26.1

18.7

0.39

29.4

20.6

0.41




Day +21

18.2

19

0.78

24

14.9

0.64




Day +28

11.8

24.6

0.23

18.6

17.4

0.16




Day +60

26.7

15.2

0.49

19.3

12.4

0.58




IL-12p70

Day -1

2.5

2.5

0.73

NA




Day +1

2.9

2.4

0.75

3.1

2.2

0.58




Day +7

1.5

2.5

0.38

2.2

2.2

0.35




Day +14

1.4

2.3

0.11

2.2

2

0.14




Day +21

1.9

2.7

0.01

2.6

2.2

0.01




Day +28

2.2

3

0.23

2.3

2.9

0.28




Day +60

2.4

2.7

0.80

2.9

2.6

0.80



Supplementary Table 4. Cytokine serum concentrations according to patient and donor age. NA: not applicable. Patient and donor’s age were reported as median value at HSCT. The differences between groups were calculated by the Mann-Whitney test.







Variables

Baseline

value

Compared value

RR

95% CI

P-value

IL-2Rα

day +14

Donor type

MFD

MUD

1.11

0.06-19.6

0.93

HSC source

Others

PB

2.3

0.67-6.92

0.13

Conditioning

RTR

MAC

0.79

0.36-1.73

0.56

Patient’s age

<9.7 y

≥9.7 y

0.93

0.46-1.87

0.84

Donor’s age

<21.9 y

≥21.9 y

0.75

0.30-1.84

0.53

IL-2Rα

day +21

Donor type

MFD

MUD

1.38

0.07-24.9

0.82

HSC source

Others

PB

2.03

0.6-6.83

0.25

Conditioning

RTR

MAC

0.84

0.37-1.90

0.69

Patient’s age

<9.7 y

≥9.7 y

1.53

0.75-3.12

0.23

Donor’s age

<21.9 y

≥21.9 y

0.60

0.24-1.44

0.25

HGF

day +14

Donor type

MFD

MUD

2.4

0.12-47

0.56

HSC source

Others

PB

3.39

1.11-10.28

0.03

Conditioning

RTR

MAC

0.95

0.38-2.36

0.91

Patient’s age

<9.7 y

≥9.7 y

1.54

0.68-3.49

0.29

Donor’s age

<21.9 y

≥21.9 y

2.74

0.89-8.43

0.07

HGF

day +21

Donor type

MFD

MUD

0.34

0.01-7.01

0.49

HSC source

Others

PB

2.21

0.6-8.14

0.23

Conditioning

RTR

MAC

0.80

0.34-1.87

0.61

Patient’s age

<9.7 y

≥9.7 y

2.25

1.09-4.65

0.02

Donor’s age

<21.9 y

≥21.9 y

3.52

1.25-9.87

0.01

IL-8

day +28

Donor type

MFD

MUD

0.007

0.002-0.02

<0.001

HSC source

Others

PB

2

0.5-8.6

0.31

Conditioning

RTR

MAC

0.75

0.75-2.29

0.61

Patient’s age

<9.7 y

≥9.7 y

0.29

0.11-0.73

<0.001

Donor’s age

<21.9 y

≥21.9 y

1.09

0.36-3.33

0.87

Supplementary Table 5. Multivariate analysis of factors affecting cytokine serum concentrations at established time-points. MFD=matched family donor, MUD=matched unrelated donor, PB=peripheral blood stem cells, MAC=myeloablative regimens, RTR=reduced intensity regimes. This multivariate analysis was performed according to multinomial logistic regression model by SPSS software.








Conditioning Regimens

1999-2002

2003-2006

2007-2009

TBI-based

26 (68%)

24 (33%)

30 (49%)

Busulfan-based

5 (13%)

21 (29%)

18 (29%)

Others

7 (18%)

26 (37%)

13 (21%)

P

0.008




GVHD Prophylaxis













Cy-A+MTX +/- sterods

7 (18%)

11 (15%)

11 (18%)

Cy-A+serotherapy ±MTX±steroids

19 (50%)

42 (59%)

45 (74%)

Cy-A

12 (31%)

18 (25%)

5 (8%)

P

0.003


Supplementary Table 6. Conditioning regimens and GVHD prophylaxis given during study period. The analysis was performed through the X2 test by the SPSS software.


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