A Next-Gen Sequencing Assay for the Simultaneous Detection of
Bladder Cancer-Associated Protein and DNA Markers
Anthony P. Shuber, CTO, Co-Founder
AACC Oak Ridge Conference
April 2013
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Agenda
• “Why” combine DNA and Protein Biomarkers
– CIDD Approach (Clinical Intervention Determining Diagnostic)
• “How” we combine DNA and Protein Biomarkers
– MADR Approach (Multiple Analyte Diagnostic Readout)
– Application to Bladder Cancer
• Simultaneous Analysis of Protein and DNA on a Next Gen
Seq Platform
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Disease Heterogeneity Creates Ambiguity
Disease Free
Cancer
80% Sens./80% Spec.
0
Biomarker
Results
20% C 20% N
1000
Standard Analytical Approach
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1
Stratification of Patient Population
Clinical Intervention Determining Diagnostic (CIDD) Approach
Disease Free
0
Biomarker
Results
No Intervention
(>90% Sen.)
Cancer
Standard
Intervention
Maximum Intervention
(>90% Spec.)
1000
High Spec.
PPV
High Sens.
NPV
4
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MADR Reduces Population Overlap
Disease Free
Cancer
MMP-2 MMP-2
<0.400ng/ml
<1.100ng/ml
DNA marker positive samples
(FGFR3)
Biomarker
0 Results
No Intervention
•
Standard
Intervention
1000
Maximum Intervention
DNA plus protein markers can result in an increase in Sensitivity and
Specificity simultaneously, maximizing NPV and PPV
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5
CIDD/MADR Application to
Triaging Hematuria Population
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2
Hematuria Triage (PBS-002) Marker Panel
MMP2
• Matrix Metalloproteinase 2 (MMP-2)
FGFR3
• Fibroblast Growth Receptor 3 (qFGFR3)
– Involved with Angiogenesis, Tissue-remodeling (Tumor Growth) and Metastasis
– Demonstrated Association with Multiple Cancers
– Quantitative (Can achieve high sensitivity)
–
–
–
–
Cell surface Receptor Tyrosine Kinase for Fibroblast Growth Factor
Binary results
High specificity
Associated with genetically stable bladder tumors of low grade and stage
• Twist1 and Nidogen2
TWIST1/NID2
– Twist1: transcription factor involved in multiple developmental pathways
– Nidogen2: basement membrane protein
– Binary or quantitative
• Performance Established in Urine
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PBS 002: Hematuria Triage Study
• 27 Clinical Sites (2 Academic, 25 Community Practices)
• Total Number of Evaluable Subjects
748
• Cancers
58
• Hematuria+/ Cystoscopy690
PBS-002 Version 1
Study
Markers
PBS-002
Cutoffs
NPV
Sensitivity
Specificity
58 cancers
690 H+/C-
FGFR3
MMP-2<1.100
Twist1 <139k
Nid2<680k
98.2%
(388/395)
[96-99%]
87.9%
(51/58)
[76-95%]
56.2%
(388/690)
[52-60%]
Patient Distribution
Negative*
Intermediate
53% (12% cancer, 56% HC)
N/A
44% (53% cancer, 44% HC)
Positive (qFGFR3 pos)
PPV
Sensitivity
Specificity
95.2%
(20/21)
[76-100%]
34.5%
(20/58)
[22-48%]
99.9%
(689/690)
[99-100%]
3% (35% cancer, .1% HC)
* all marker negative for FGFR3, MMP-2, Twist1 and Nid2
Karnes et al., Mayo Clinic Proceedings, 2012;87(9):835-42.
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Application of Next Gen Sequencing
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3
Next Gen Sequencing of FGFR3 Increases Sensitivity in Urine
• 43 urine samples from cancer patients were tested by ngsFGFR3
Sensitivity
Stage
qPCR
Deep Sequencing
Ta
11.1%
(3/27)
63.0%
(17/27)
T1
22.2%
(2/9)
55.6%
(5/9)
≥T2
0.0%
(0/7)
28.6%
(2/7)
All
Stages
11.6%
(5/43)
[5-24%]
55.8%
(24/43)
[40-71%]
• 5 samples were previously positive by qPCR. All were positive by
ngsFGFR3.
• ngsFGFR3 detected an additional 19 cancers that are as low as 0.02%
mutant
Millholland et al., Research and Reports in Urology, 2012;4(1):33-40.
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TP53 Complements FGFR3 Sensitivity in Bladder Cancer
•
TP53 mutations found in ~30% of all bladder tumors
•
TP53 mutations have very little overlap with FGFR3 mutations
•
•
Stage: pTa: 18%, pT1: 47%, ≥pT2: 52%
Grade: Low: 19%, High: 51%
noninvasive
invasive
van Rhijn et al. JCO May 2003 and Sjodahl et al. PLoS1 6(4):e18583, 2011
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ngsTP53 Increases Clinical Sensitivity
•
57 tissues were analyzed both for ngsFGFR3 and ngsTP53
•
17 Ta, 21 T1, 3 Tis, 16 ≥T2
Stage
ngsFGFR3
ngsTP53
ngsFGFR3/ngs
TP53
Ta
10/17 (59%)
1/17 (6%)
11/17 (65%)
T1
8/21 (38%)
1/21 (5%)
9/21 (43%)
Tis
0/3 (0%)
1/3 (33%)
1/3 (33%)
≥T2
2/16 (13%)
8/16 (50%)
10/16 (63%)
Total
20/57 (35%)
11/57 (19%)
31/57 (54%)
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Independent MADR Workflow
MMP ELISA Workflow
FGFR3/TP53 NGS Workflow
Urine Sample Incubation
DNA Prep
Wash
Conjugate Addition + Incubation
FGFR3/TP53 Multiplex Primary
PCR (7 exons, 1rx)
Wash
Substrate Solution Addition + Incubation
FGFR3/TP53 Mutant
Analysis (142 Mutations)
Read Optical Density
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Platform-Agnostic MADR Technology
Single Molecule Sequencing MADR Workflow
Protein (Urine)
DNA Prep
Aptamer
Selection
Sequence –
specific
amplification
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Protein Marker Detection by NGS (Model System)
MMP-2 Detection by ELISA
MMP-2 Detection by qPCR
MMP-2 Detection by NGS
blue: 6.25ng/ml MMP-2
red: 3.125ng/ml MMP-2
green: 1.56ng/ml MMP-2
pink: 0.78ng/ml MMP-2
grey: 0.39ng/ml MMP-2
*All analyses done in triplicate
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Protein Marker Detection by NGS (Urine)
MMP-2 Detection by ELISA
R² = 0.9894
MMP-2 Detection by qPCR
MMP-2 Detection by NGS
R² = 0.9745
R² = 0.9593
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Simultaneous Protein and DNA Detection by NGS
Sequencing Reads
Single Marker Analysis
MMP-2/Aptamer
600000
500000
ngsFGFR3
400000
300000
Sample
1
2
3
4
5
200000
% Mutant
0%
0%
0%
0%
0%
6
7
48.3% 3.6%
8
3.6%
9
10
4.5% 10.8%
100000
0
1
2
3
4
5
6
7
8
9
10
Sample
MMP-2/Aptamer +
ngsFGFR3
Sequencing Reads
Multiplex Protein/DNA Marker Analysis
200000
150000
100000
50000
0
1
2
3
4
5
6
7
8
9
10
Sample
Sample
1
2
3
4
5
% Mutant
0%
0%
0%
0%
0%
6
7
49.3% 2.4%
8
9
10
2.3%
3.2%
7.9%
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Conclusions
• Combining Protein and DNA markers in a single assay
improves clinical performance
• Application of NGS increases analytical and clinical
sensitivity
• NGS associated protein and DNA analysis reduces assay
complexity and reduces cost
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Acknowledgements
•
•
•
•
•
•
•
•
•
•
Cecilia Fernandez
John Millholland
Andrew Dunn
Autumn Duchesne
Lydia Anderson
Maria Campo
Maria Muraca
Carol Ahearn
Holly Gettler
Alisha Josey
• Jeff Karnes
– Mayo Clinic
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