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SJIA PLASMA BIOMARKER DISCOVERY STUDY DESIGN. DIGE analysis. Cluster analysis. ELISA assays. Prediction analysis (LDA). Two class classification. 1. 3. 2. Predictor discovery in training set. Predictor test in testing set. DIGE raw gel images SJIA ( 10 F , 10 Q) POLY (5 F, 5 Q). - PowerPoint PPT Presentation
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Classifier training
Mann Whitney
Predictor discoveryin training set
4
Training setSJIA (12 F, 12 Q)POLY (13 F, 10 Q)
1
DIGEraw gel imagesSJIA (10 F, 10 Q)POLY (5 F, 5 Q)
Spot findingspot alignment
feature extraction
889 discrete spot features
Classifier training
ClassifySJIA F vs QPOLY F vs Q
DIGE analysis Prediction analysis (LDA)
Predictor testin testing set
5
Testing setSJIA (10 F, 10 Q)POLY (10 F, 5 Q)
2
Cluster analysis
2d hierarchicalclustering
heatmap plotting
Normalizationmanual review
Manual review
MSMS ID96 spots
3
8 proteincandidates
Assay development
7 ELISA assays
7
DiscriminateSJIA F
KDFI
SJIA PLASMA BIOMARKER DISCOVERY STUDY DESIGN
ClusteringBox-and-Whisker Analysis
DiscriminateSJIA FSJIA QPOLY FPOLY Q
ELISA assays
ELISA assay
ELISA assayFC (27 )KD (10)
Two class classification
Classification analysis
9
ELISA assayQ->F (11)Q->Q (14)
Test to anticipate SJIA F in Q
LDAFisher exact test
P < 10-5
randomization
Blind testing
ClassifySJIA F vs QPOLY F vs Q
Fisher exact test
Training setSJIA F(12) KD (7), FC (15)
ClassifySJIA F vs Non SJIA F
Testing setSJIA F(10) KD (3), FC (12)
randomization
Blind testing
ClassifySJIA F vs Non SJIA F
Fisher exact test
8
Two dimensional DIGE analysis identified 96 protein spots differentially expressed between SJIA flare and quiescence
A B CF Q F Q
SJIA POLY
F Q
SJIA
F Q
POLY
DIGE analysis reveals a seven protein biomarker panel in plasma clearly differentiating SJIA flare from quiescence
B C DF Q F Q
SJIA POLY
F Q
SJIA
F Q
POLY
A2M
APOA1
SAP
CRP
HP
MRP14
SAAMRP8
A2M
APOA1
SAP
CRP
HP
MRP14
SAAMRP8
F Q F QSJIA POLY
APOA1 SAPCRP HP MRP14 SAAMRP8
Relative expression
2
3
4
1
0
A2M
A
Training setn = 24
12 12
Clinicaldiagnosis F Q
n =
LDA
11 1
1 11
Classified as F
Classified as Q
PercentAgreementwith clinical
diagnosis
91.6% 91.6%+ -
91.6%
Overall
P = 1.0 X 10-3
Testing setn = 20
10 10
Clinicaldiagnosis F Q
n =
Testing
8 3
2 7
Classified as F
Classified as Q
PercentAgreementwith clinical
diagnosis
80% 70%+ -
75%
Overall
P = 7 X 10-2
B CA
Biomarker panelof 7 members
1. A2M
2. APO AI
3. CRP
4. HP
5. MRP8/MRP14
6. SAA
7. SAP
SJIA SJIA
DTraining
SJIA F SJIA Q SJIA F SJIA Q
Testing
Pred
icte
d pr
obab
ilitie
sPatient samples
E
Sen
sitiv
ity
1- Specificity
CRP : AUC=0.58
SJIA F vs. Q
panel : AUC=0.82
ESR : AUC=0.86
ELISA analysis validates the seven protein biomarker panel in plasma
Training setn = 23
13 10
Clinicaldiagnosis F Q
n =
LDA
10 5
3 5
Classified as F
Classified as Q
PercentAgreementwith clinical
diagnosis
76.9% 50%+ -
65.2%
Overall
P = 0.41
Testing setn = 15
10 5
Clinicaldiagnosis F Q
n =
Testing
3 0
7 5
Classified as F
Classified as Q
PercentAgreementwith clinical
diagnosis
30% 100%+ -
53.3%
Overall
P = 0.20
A B
POLY POLY
CTraining
PF PQ PF PQ
Testing
Pred
icte
d pr
obab
ilitie
sPatient samples
D
1- Specificity
CRP : AUC=0.64
POLY F vs. Q
panel : AUC=0.64
Sen
sitiv
ity
ELISA analysis invalidates the seven protein biomarker panel in POLY plasma
Training setn = 25
11 14
Clinicaldiagnosis QF QQ
n =
LDA
6 2
5 12
Classified as QF
Classified as QQ
PercentAgreementwith clinical
diagnosis
54.5% 85.7%+ -
72%
Overall
P = 0.08
A
SJIA
B
Pred
icte
d pr
obab
ilitie
s
Patient samples
C
CRP : AUC=0.59
SJIA QF vs. QQ
panel : AUC=0.78
ESR : AUC=0.60
Training
QQ QF
Sen
sitiv
ity
1- Specificity
ELISA analysis shows the ineffectiveness of seven protein plasma biomarker panelin prognosis of impending SJIA flare
B C
MRP14
F KD FC
SJIA
A2M
APOA1
SAP
CRP
HP
SAAMRP8
1210 12
SJIAF KD FCData set
n = 34
10
Clinicaldiagnosis
SJIAF
NOT-SJIAF
n =
Unsupervised clustering
7
3
1
23
Clustered as SJIA F
Clustered as NOT-SJIA F
PercentAgreementwith clinical
diagnosis
70% 95.8%+ -
88.2%
Overall
P = 1.6 X 10-4
24
Prot
ein qu
antity
F KD FCSJIA
APOA1 SAPCRP HP MRP14 SAAMRP8A2M
2
3
4
1
0
A
DIGE analysis shows that seven protein SJIA flare panel in plasma clearly differentiating SJIA flare from confounding Kawasaki and febrile illness
Training setn = 34
12 22
Clinicaldiagnosis
n =
LDA
12 0
0 22
PercentAgreementwith clinical
diagnosis
100% 100%+ -
100%
Overall
P = 7.4X 10-7
Testing setn = 25
10 15
Clinicaldiagnosis
n =
Testing
9 0
1 15
PercentAgreementwith clinical
diagnosis
90% 100%+ -
93.3%
Overall
P = 4.9 X 10-6
A B
SJIAF
C
Training
FC
Testing
Pred
icte
d pr
obab
ilitie
s
Patient samples
712 15
SJIAF KD FC
NOTSJIA F
SJIAF
NOTSJIA F
310 12
SJIAF KD FC
KD SJIA F FC KD SJIA F
Clustered as SJIA F
Clustered as NOT SJIA F
ELISA analysis validates the utility of the seven protein SJIA flare panel in plasma to discriminate SJIA flare from confounding Kawasaki and febrile illness
Training setn = 39
16 23
Clinicaldiagnosis F Q
n =
LDA
11 3
5 20
Classified as F
Classified as Q
PercentAgreementwith clinical
diagnosis
68.8% 87%+ -
79.5%
Overall
P = 6.0X 10-4
Bootstrap setn = 73
34 39
Clinicaldiagnosis F Q
n =
Testing
29 7
5 32
Classified as F
Classified as Q
PercentAgreementwith clinical
diagnosis
85.3% 82.1%+ -
83.5%
Overall
P = 5.9 X 10-9
B C
SJIA SJIA
DTraining
SF SQ SF SQ
Bootstrap confirmation
Pred
icte
d pr
obab
ilitie
sPatient samples
E
1- Specificity
Bootstrap : AUC=0.90
SJIA F vs. Q
Training : AUC=0.84
Sen
sitiv
ity
A
Biomarker panelof 7 members
1. TIMP1
2. MMP9
3. IL18
4. RANTES
Agilent protein array analysis reveals a four protein SJIA flare panel in plasma clearly differentiating SJIA flare from quiescence
Training setn = 13
6 7
Clinicaldiagnosis F Q
n =
LDA
5 1
0 6
Classified as F
Classified as Q
PercentAgreementwith clinical
diagnosis
100% 85.7%+ -
84.6%
Overall
P = 0.01
Bootstrap setn = 16
6 10
Clinicaldiagnosis F Q
n =
Testing
5 2
1 8
Classified as F
Classified as Q
PercentAgreementwith clinical
diagnosis
83.3% 80%+ -
81.25%
Overall
P = 0.03
A B
POLY POLY
CTraining
PF PQ PF PQ
Pred
icte
d pr
obab
ilitie
sPatient samples
D
1- Specificity
Bootstrap: AUC=0.91
POLY F vs. Q
Training: AUC=1
Sen
sitiv
ity
A
Biomarker panelof 4 members
1. TIMP2
2. IGFBP-3
3. IGFBP-6
4. VCAM-1
Agilent protein array analysis reveals a four protein POLY flare panel in plasma clearly differentiating POLY flare from quiescence
Bootstrap confirmation