SJIA flare signature analysis

2

Discovery set(ND.SAF vs. AF.QOM.RD.KD.FI)

1

LCMSraw spectra

Peak findingpeak alignment

feature extraction

Urine peptide

index

NSC feature selection

Ten-foldCross-validation

Urine profiling Urine biomarker analysis (NSC, LDA, ROC)

Featureselection

Identification of FGA peptides

LDA analysis

6 peptide biomarker panel

ROC analysis500 bootstrap samples

3

Classification

ND.SAF vs.

KD.FI

ND.SAF vs.

QOM.RD

SJIA flare signature analysis

4

Discovery set(SAF, QOM)

NSC feature selection

Ten-foldCross-validation

Feature selection

LDA analysis

6 protein biomarker panel

ROC analysis

5

Classification

Training Samples

SAF vs.

QOM

“bootstrap” samples

SAF vs.

QOM

Literaturereview

Antibody arrayconstruction

Hypothesisgeneration

Cherry pick43 protein antibodies

Antibody array assay

Antibody arrayprofiling

Plasma biomarker analysis (NSC, LDA, ROC)

6

FIGURE 1

patient demographics obs. mean median s.d. min max obs. mean median s.d. min max obs. mean median s.d. min max obs. mean median s.d. min max obs. mean median s.d. min max1 Age 2 3 3 2.83 1 5 18 11.72 12.5 4.5 3 17 9 13 13 1.66 11 16 18 12.44 13 4.09 5 17 9 12.44 14 5.57 6 212 Sys score 2 2.5 2.5 0.71 2 3 18 1.61 2 0.61 1 3 8 0.12 0 0.35 0 1 18 0 0 0 0 0 8 0 0 0 0 03 Arth poly score 1 0 0 NA 0 0 11 1.55 1 0.69 1 3 6 1.33 1 0.52 1 2 12 0 0 0 0 0 5 0 0 0 0 04 Arth Sys score 2 1 1 0 1 1 18 2.78 3 1.11 0 4 8 2.62 2 0.92 2 4 18 0.67 1 0.49 0 1 8 0.62 1 0.52 0 15 Joint Ct 2 0 0 0 0 0 18 9.78 6 11.22 0 40 8 5.75 3.5 4.77 2 15 18 0 0 0 0 0 8 0 0 0 0 06 Fever 2 1 1 0 1 1 18 0.44 0 0.51 0 1 8 0 0 0 0 0 18 0 0 0 0 0 8 0 0 0 0 07 Rash 2 1 1 0 1 1 18 0.5 0.5 0.51 0 1 8 0 0 0 0 0 18 0 0 0 0 0 8 0 0 0 0 08 ESR 2 54 54 8.49 48 60 16 51.94 47.5 27.36 5 108 7 9.29 9 5.19 0 17 17 9.53 5 9.06 0 29 6 4.83 5.5 1.47 3 69 d dimers 1 5433 5433 NA 5433 5433 17 1887.94 1500 1139.69 200 4403 5 437.51 220 416.24 0.55 900 7 693 900 318.84 220 900 3 666.67 900 404.15 200 900

10 CRP 1 29.4 29.4 NA 29.4 29.4 13 28.05 12.2 28.96 1.8 82.7 5 1.96 0.2 3.72 0.2 8.6 8 1.81 0.8 3.22 0.2 9.7 4 0.45 0.3 0.38 0.2 111 Ferritin 2 1549.5 1549.5 224.15 1391 1708 16 654.62 203.5 877.07 26 3441 7 30.22 38.6 19.26 7.28 53 17 34.76 19 29.88 8 103 5 22.4 20 11.78 11.2 38.112 WBC 2 14.6 14.6 3.96 11.8 17.4 18 15.13 13.05 7.87 4.3 38.5 8 9.41 7.25 4.41 5.9 18.7 17 6.19 5.9 1.36 4.2 8.4 5 6.12 5.6 2.07 4.1 9.613 PLT 2 480.5 480.5 102.53 408 553 18 514.94 492 168.99 226 829 8 342.88 338 112.79 222 520 17 319.12 333 61.87 216 426 5 314.4 301 30.62 292 36714 CBC Monocyte 2 0.44 0.44 0.51 0.08 0.8 16 0.7 0.67 0.28 0.26 1.2 8 0.95 0.6 1.08 0.3 3.6 14 0.49 0.5 0.14 0.29 0.8 5 0.39 0.4 0.14 0.26 0.615 CBC Lymphocytes 2 3.28 3.28 2.35 1.61 4.94 16 2.17 2.06 1.78 0.06 7.92 8 2 1.9 1.18 0.2 4.26 14 1.97 2.02 0.63 0.92 2.9 5 2.32 2.1 0.86 1.68 3.7916 CBC Neutrophils 2 10.68 10.68 0.9 10.04 11.31 16 12.15 10.24 7.83 3.41 35.6 8 6.34 4.75 4.36 3 16.5 14 3.28 2.8 1.59 1.36 6.8 5 3.25 2.9 1.16 2.09 5.1518 Treatment intensity score 2 1 1 0 1 1 16 2.75 2.75 1.66 1 6 8 2.38 1.5 2.13 0 6 13 2.77 3 1.09 1 4 7 0 0 0 0 019 Gender (male%) 2 18 9 17 1020 NSAID % 2 16 8 13 821 PO.PRED % 2 16 8 13 822 MTX % 2 17 8 13 823 TNF % 2 16 8 13 824 IL.1.RA % 2 17 8 13 8

100% 33% 33% 39% 22%

ND SAF AF QOM RD

0% 50% 38% 29% 0%100% 88% 62% 86% 0%

0% 12% 25% 50% 0%0% 47% 50% 71% 0%

0% 35% 12% 36% 0%

TABLE 1

num. obs. mean median s.d. min max num. obs. mean median s.d. min maxage 23.00 3.52 3.00 2.31 1.00 10.00 23.00 3.91 2.00 3.82 1.00 16.00

illDay 23.00 7.74 7.00 4.06 2.00 22.00 23.00 5.91 5.00 4.82 0.00 22.00wbc 22.00 12.74 12.45 4.75 5.90 26.80 19.00 9.73 9.70 3.83 3.90 17.00

polys 23.00 46.93 46.00 15.39 7.00 75.00 19.00 48.37 49.00 16.38 5.00 76.00bands 23.00 12.13 8.00 12.31 0.00 38.00 15.00 8.13 6.00 7.19 0.00 23.00

lymphs 23.00 29.47 20.00 19.15 7.00 82.00 19.00 32.90 30.00 13.57 12.00 63.00monos 23.00 6.46 6.00 3.65 3.00 17.00 19.00 7.22 7.00 3.69 1.00 14.00

eos 23.00 2.11 2.00 2.34 0.00 8.00 19.00 0.89 0.00 2.26 0.00 10.00hgb 23.00 11.32 11.30 0.94 9.40 13.00 19.00 11.96 11.90 1.04 9.60 13.80hct 23.00 33.02 32.40 2.86 27.10 38.10 19.00 35.30 35.50 2.94 28.60 41.00esr 23.00 51.26 45.00 28.05 5.00 140.00 12.00 30.67 34.50 22.90 0.00 81.00crp 21.00 7.40 5.00 6.10 0.30 21.20 13.00 3.39 2.10 3.51 0.30 12.00plts 22.00 400.68 404.50 103.07 236.00 642.00 19.00 298.84 267.00 102.08 144.00 585.00alt 20.00 113.60 60.50 138.63 14.00 510.00 12.00 42.25 22.50 53.14 7.00 166.00ggt 21.00 73.67 34.00 68.95 10.00 222.00 8.00 22.38 14.50 26.77 6.00 88.00ua 19.00 30.63 20.00 49.51 0.00 226.00 16.00 12.81 6.50 14.99 2.00 55.00

gender (male%) 23.00 23.00

KD FI Clinical parameters

81.82 60.87

TABLE 2

FGA(20-35) 1536.61 ADSGEGDFLAEGGGVRFGA(607-622) 1639.77 AGSEADHEGTHSTKRG

FGA(605-621) 1826.80 DEAGSEADHEGTHSTKR FGA(605-622) 1883.80 DEAGSEADHEGTHSTKRG FGA(605-628) 2560.2 DEAGSEADHEGTHSTKRGHAKSRP FGA(605-629)* 2659.24 DEAGSEADHEGTHSTKRGHAKSRPV

MH+Protein Sequence

Relative abundance

ND.SAF AF.QOM.RD.KD.FI

0.00150.09860.32250.6016 0.00370.1443

-0.0004-0.024-0.0787-0.1467-0.0009-0.0352

TABLE 3

FGA(605-621)1826.80

FGA(605-622)1883.80

FGA(605-629)*2659.24

SAF AFQOM RD KD FI HCND

FGA(607-622)1639.77

FGA(605-628)2560.2

FGA(20-38)1536.69

SAF AFQOM RD KD FI HCND

FGA peptide marker distribution

Relative abundance

FIGURE 2

Classification

2+18 23+23

Clinicaldiagnosis

ND.SAF KD.FI

n =

LDA

14 1

6 45

Predicted as SJIA F

Predicted as non SJIA F

PercentAgreementwith clinicaldiagnosis

70% 97.8%+ -

89.4%

Overall

P = 6.7X10-9

A B

ND SAF KD FI

Pre

dic

ted

pro

bab

ilit

ies

Patient samples

Sen

siti

vity

1- Specificity

Mean(AUC): 95.6%

C

FIGURE 3

Classification

2+18 18+9

Clinicaldiagnosis

ND.SAF QOM.RD

n =

LDA

13 2

7 25

Predicted as SJIA F

Predicted As SJIA Q

PercentAgreementwith clinicaldiagnosis

65% 92.6%+ -

80.9%

Overall

P = 6.1X10-5

A B

ND SAF QOM RD

Pre

dic

ted

pro

bab

ilit

ies

Patient samples

Sen

siti

vity

1- Specificity

Mean(AUC): 91.0%

C

FIGURE 4

       MH+ Sequence FGA location  

1465.58 DSGEGDFLAEGGGVR 21-35  1536.61 ADSGEGDFLAEGGGVR 20-35  1775.74 DSGEGDFLAEGGGVRGPR 21-38  1846.77 ADSGEGDFLAEGGGVRGPR 20-38         1339.68 SQLQKVPPEWK 239-249  1252.65 QLQKVPPEWK 240-249         1686.71 GGSTSYGTGSETESPRN 272-288  2473.02 GGSTSYGTGSETESPRNPSSAGSWN 272-296         2375.07 GSTGNRNPGSSGTGGTATWKPGSSGP 303-328  1360.61 PGSSGTGGTATWKPG 310-324         1554.66 DGFRHRHPDEAAF 507-519         2553.01 SSSYSKQFTSSTSYNRGDSTFES 576-598  2768.13 SSSYSKQFTSSTSYNRGDSTFESKS 576-600  2931.15 SSSYSKQFTSSTSYNRGDSTFESKSY 576-601  1913.75 QFTSSTSYNRGDSTFES 582-598         1354.52 DEAGSEADHEGTH 605-617  1542.60 DEAGSEADHEGTHST 605-619  2020.90 DEAGSEADHEGTHSTKRGH 605-623  2091.92 DEAGSEADHEGTHSTKRGHA 605-624  2560.20 DEAGSEADHEGTHSTKRGHAKSRP 605-628  2659.24 DEAGSEADHEGTHSTKRGHAKSRPV 605-629  

2344.12 GSEADHEGTHSTKRGHAKSRPV 608-629  

2730.26 ADEAGSEADHEGTHSTKRGHAKSRPV 604-629  2293.96 MADEAGSEADHEGTHSTKRGHA 603-624  2762.25 MADEAGSEADHEGTHSTKRGHAKSRP 603-628  2861.28 MADEAGSEADHEGTHSTKRGHAKSRPV 603-629  2877.31 MADEAGSEADHEGTHSTKRGHAKSRPV* 603-629  2122.81 SYKMADEAGSEADHEGTHST 600-619  2672.11 SYKMADEAGSEADHEGTHSTKRGHA 600-624  3239.46 SYKMADEAGSEADHEGTHSTKRGHAKSRPV 600-629  3255.46 SYKMADEAGSEADHEGTHSTKRGHAKSRPV* 600-629  

851.49 GHAKSRPV 622-629  

                     

I

II

III

IV

V

VI

VII

TABLE 4

B D Training Bootstrapping testingA

FIGURE 5

SAF QOM

Pre

dic

ted

pro

ba

bili

tie

s

Patient samples

SAF QOM

Training samplesn = 39

25 14

Clinicaldiagnosis F Q

n =

LDA

23 4

2 10

Classified as F

Classified as Q

PercentAgreementwith clinical

diagnosis

92% 71.4%+ -

84.6%

Overall

P = 7.9X 10-5

Bootstrapping samplesn = 52

41 11

Clinicaldiagnosis F Q

n =

Testing

36 2

5 9

Classified as F

Classified as Q

PercentAgreementwith clinical

diagnosis

87.8% 81.8%+ -

86.5%

Overall

P = 2.4 X 10-5

C

SJIA SJIA

                Relative abundance  

biomarkers SAF QOM  TIMP-1 0.2782 -0.4967  IL-18 0.1735 -0.3099  

RANTES 0.1681 -0.3002  P-Selectin 0.1616 -0.2885  MMP-9 0.1308 -0.2335  L-Selectin 0.0121 -0.0216  

       

Sen

siti

vity

1- Specificity

Mean(AUC): 92.2%

Sen

siti

vity

1- Specificity

Mean(AUC): 90.7%

Training Bootstrapping testing

FIGURE 6

A C Training Bootstrapping testing

FIGURE 5

SAF QOM

Pre

dic

ted

pro

ba

bili

tie

s

Patient samples

SAF QOM

Training samplesn = 47

LDA

13 2

7 25

Classified as F

Classified as Q

PercentAgreementwith clinical

diagnosis

65% 92.6%+ -

80.9%

Overall

P = 6.1X 10-5

Bootstrapping samplesn = 24

15 9

Clinicaldiagnosis F Q

n =

Testing

7 1

8 8

Classified as F

Classified as Q

PercentAgreementwith clinical

diagnosis

87.8% 81.8%+ -

86.5%

Overall

P = 2.4 X 10-5

B

SJIA

Clinicaldiagnosis

ND.SAF QOM.RD

n =

SJIA

2+18 18+9

Sen

siti

vity

1- Specificity

Mean(AUC): 90.8%

Sen

siti

vity

1- Specificity

Mean(AUC): 80.9%

Training Bootstrapping testing

FIGURE 6