RNA biomarkers enable early disease detection, assessment of prognosis, monitoring patient response to therapy or selecting those treatments most likely to be efficacious for a given patient. As such, biomarker discovery is a promising approach toward effective precision medicine, especially when applied to body fluids that allow for minimally invasive testing. Biogazelle has developed a 7-steps approach covering all aspects from biomarker discovery to validation of qPCR based biomarker tests. RNA sequencing methods were optimized for performance on a clinically relevant low input samples such as serum or FFPE. After data processing by our dedicated Cobra pipeline, advanced statistical modeling is applied to generate a set of candidate biomarker panels. Based on >15 years of experience in qPCR assay design and validation, a qPCR based biomarker test is developed and validated analytically and clinically. As an example we will show how small RNA sequencing on serum samples was successfully used for the development of a treatment selection biomarker in metastatic prostate cancer.

1. © 2017 Biogazelle. All rights reserved. 1
Using the potential of RNA in body
fluids for biomarker development
Jan Hellemans, CEO Biogazelle
Knowledge for Growth, Ghent, Belgium
May 18, 2017

2. © 2017 Biogazelle. All rights reserved. 2
“A biomarker, or biological marker, generally
refers to a measurable indicator of some
biological state or condition”
diagnosis
monitoring
prognosis
safety
prediction
stratification

3. © 2017 Biogazelle. All rights reserved. 3
Low clinical development success rate
63%
30%
57%
85%
9%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
phase 1 > phase 2 phase 2 > phase 3 phase 3 > NDA/BLA NDA/BLA >
approval
phase I > approval
Series2
source: Amplion

4. © 2017 Biogazelle. All rights reserved. 4
Biomarkers increase likelihood of
approval 3x
77%
47%
77%
95%
26%
63%
29%
55%
84%
8%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
phase 1 > phase 2 phase 2 > phase 3 phase 3 > NDA/BLA NDA/BLA >
approval
phase I > approval
with selection biomarker without biomarker
source: Amplion

5. © 2017 Biogazelle. All rights reserved. 5
In 2015, 28% of NNDs approved by FDA
were precision medicines
0
500
1000
1500
2000
2500
1985 1990 1995 2000 2005 2010 2015 2020 2025
Trialswithbiomarkers
measured
projected
source: Amplion

6. © 2017 Biogazelle. All rights reserved. 6
advantages of liquid
biopsies
nucleic acid
biomarkers
serial sampling minimally invasive
reduced risk easy
patient comfort faster sampling
mRNA
miRNA
lncRNA
DNA

7. © 2017 Biogazelle. All rights reserved. 7
Biomarker development program
biomarker
discovery
qPCR test
development
test validation

8. © 2017 Biogazelle. All rights reserved. 8
Biomarker development program
biomarker
discovery
qPCR test
development
test validation
• expression profiling
• feasibility study
• model development
70%
93%
70%
93%
70%
93%

9. © 2017 Biogazelle. All rights reserved. 9
Case studies
prostate cancer endometriosis
• dr. Piet Ost
• Ghent University Hospital
• oncology
• treatment stratification
• dr. Arne Vanhie
• UZ Leuven
• gynaecology
• diagnostic test

10. © 2017 Biogazelle. All rights reserved. 10
Case studies
prostate cancer endometriosis
• cohort 1: 40 patients
• serum samples
• small RNA sequencing
• 10-20M reads > ~600 miRNAs
• cohort 1: 120 patients
• plasma samples
• small RNA sequencing
• 10-30M reads > ~650 miRNAs
biomarker discovery – expression profiling
• optimized lab procedures for low input liquid biopsies (0.2 ml)
• ISO 17025 accredited
• Cobra analysis

11. © 2017 Biogazelle. All rights reserved. 11
Case studies
prostate cancer endometriosis
• REED analysis showed potential
• multiple significant genes
• AUC: 90% [77% - 100%]
• REED analysis showed potential
• multiple significant genes
• AUC: 97% [91% - 100%]
biomarker discovery – feasibility study
• REED: “Rapid and Easy Evaluation of Datasets”
• partnership

12. © 2017 Biogazelle. All rights reserved. 12
Case studies
prostate cancer endometriosis
•
• 50 miRNAs differ significantly
• best model has 14 miRNAs and an
AUC of 86%
• consensus list of 100 miRNAs
• statistical analysis by customer
• different comparisons
• miRNAs/model: 6-11
• AUC: 0.85-0.92
• consensus list of 42 miRNAs
biomarker discovery – model development
• advanced mathematical model building – univariate & multivariate
• other (clinical) parameters can also be integrated in the biomarker

13. © 2017 Biogazelle. All rights reserved. 13
Biomarker development program
biomarker
discovery
qPCR test
development
test validation
• assay development
• expression analysis
• retrain models
• expression profiling
• feasibility study
• model development
70%
93%
70%
93%
70%
93%
70%
93%

14. © 2017 Biogazelle. All rights reserved. 14
Case studies
prostate cancer endometriosis
• samples: identical to discovery
• geNorm: 3 reference miRNAs
• qPCR: 41 miRNAs of interest
• model with 10 miRNAs and an
AUC of 83.5% ( )
• samples: identical to discovery
• geNorm: 3 reference miRNAs
• qPCR: 41 miRNAs of interest
qPCR test development
• extensive experience in PCR assay development (PrimePCR)
• ISO 17025 accredited
• qbase+ analysis (geNorm, expression analysis & univariate statistics)

15. © 2017 Biogazelle. All rights reserved. 15
Biomarker development program
biomarker
discovery
qPCR test
development
test validation
• expression analysis
• model validation
• assay development
• expression analysis
• retrain models
• expression profiling
• feasibility study
• model development
70%
93%
70%
93%
70%
93%
70%
93%
70%
93%

16. © 2017 Biogazelle. All rights reserved. 16
Case studies
prostate cancer endometriosis
• cohort 2: 83 samples
• qPCR for the same miRNAs
• good predictive performance
could not be confirmed
• cohort 2: 90 samples
• qPCR for the same miRNAs
• different comparisons confirmed
in 2nd cohort with AUC: 0.76-0.98
biomarker validation

17. © 2017 Biogazelle. All rights reserved. 17
Biomarker development program
biomarker
discovery
qPCR test
development
test validation
• expression analysis
• model validation
• assay development
• expression analysis
• retrain models
• expression profiling
• feasibility study
• model development
70%
93%
70%
93%
70%
93%
70%
93%
70%
93%

18. © 2017 Biogazelle. All rights reserved. 18
RNA biomarker discovery and diagnostics
Expertise in different therapeutic areas and phases, some examples
indication D TR S biomarker discovery biomarker validation
clinical assay
development
clinical trial
arthritis
heart disease
endometriosis
heart disease
breast cancer
arthritis
colon cancer
T-cell lymphoma
prostate cancer
colon cancer
inflammatory bowel disease
arthritis
arthritis
multiple sclerosis
* D (diagnosis), TR (therapy response), S (stratification)

19. © 2017 Biogazelle. All rights reserved. 19
Summary
www.biogazelle.com/knowledge-center/presentations
• Biomarkers have multiple uses and may help increase the success rate
of drug development
• (non-coding) RNA from liquid biopsies is a great source of biomarkers
• Biogazelle integrates
• lab work with data analysis and statistics
• screening and validation technologies (technology switch)
• scientific excellence with quality assurance (ISO 17025 & GCLP)
• Biogazelle can assist in all stages of biomarker development
and run your biomarkers in a clinical trial or as a diagnostic test
contact us (info@Biogazelle.com) to discuss your biomarker needs

20. © 2017 Biogazelle. All rights reserved. 20
email: info@biogazelle.com
www.biogazelle.com