Poster # 1943
A microRNA signature
in urinary exosomes
for diagnosis of
prostate cancer
Thorarinn Blondal1, Anne I. Rasmussen1, Anni R. Thomsen1,
Michael Borre2, Jacob Fredsøe3, Ditte Andreasen1, James Catto4,
Torben Falck Ørntoft3, Karina D. Sørensen3 and Peter Mouritzen1.
1
4
Exiqon A/S, Vedbaek, Denmark, Depts of 2Urology and 3Molecular Medicine, Aarhus University Hospital, Denmark,
Department of Oncology, The Medical School, Sheffield, UK
Presented at the AACR conference, April 2016
Introduction
Improved diagnostic tests for prostate
cancer are needed
MicroRNAs as non-invasive
biomarkers
• Current practice: antibody based
detection of circulating prostate
specific antigen (PSA) in blood
• High rate of false positives
and negatives
• Diagnosis requires invasive FNA
(Fine-Needle Aspiration) biopsies
• MicroRNAs are stable in a range
of biofluids
• Involved in many diseases, including
roles as oncogenes and tumor
suppressors in cancer
• Used as diagnosis, prognosis, treatment
response and safety biomarkers
A non-invasive test with improved
specificity is urgently needed
microRNAs are excellent
non-invasive biomarkers for a range
of diseases
Exosomes stabilize and transfer microRNAs between cells
Exosomes
Early Endosome
MVB
ILV’s
MVB
• Exosomes are nanovesicles 40 - 140 nm in diameter
• Actively released by a wide range of cell types to the
extracellular milieu under normal and pathological conditions
• Present in a wide range of biofluids
• Carry genetic information from the cell of origin, including
microRNA
i
Golg
Lyosome
Nucleus
Figure 1.
Urinary exosomes: new liquid biopsies for cancer
• Exosomes from neoplastic cells carry potentially arrays of oncogenic
molecules including proteins and microRNAs
• The unique exosomal microRNA signature may reveal the cell of origin
and the condition of those cells
Promising non-invasive microRNA biomarkers for early
detection of malignancy
Challenges and Solutions for analysis of microRNAs in Biofluids
Limited amount
of microRNA
• Optimized exosome isolation kit to enhance microRNA signals from dilute biofluid samples
• Optimized RNA isolation kit for biofluids ensures high qPCR performance and reproducibility
• Highly sensitive qPCR detection system
MicroRNA are
challenging targets
• Short (19-22 nt) microRNAs accurately detected using short, highly specific LNA™ qPCR primers
• Robust detection of all microRNAs regardless of GC content - enabled by LNA™
• Discrimination between highly similar microRNA family members - enabled by LNA™
Undesired
components
e.g. PCR Inhibitors
Pre-analytical
variables
• RNA spike-ins to monitor RNA isolation efficiency and co-purification of inhibitors
• Optimal experimental design (biological replicates)
• Control sources of technical variation e.g. collection sites
Figure 2.
Methods
Technologies to enable microRNA biomarker analysis in liquid biopsies
Sample
Exosome
Isolation
3 ml cell-free urine
miRCURY™ Exosome
Isolation Kit - Cells, urine and CSF
RNA Isolation
miRCURY™ RNA
Isolation Kit – Cell & Plant
microRNA
quantification
miRCURY LNA™ Universal RT
microRNA PCR
Figure 3.
Exiqon has developed technologies to fulfil these key requirements:
• Methods suitable for clinical liquid biopsies (serum, plasma, urine, CSF etc.) collected using standard protocols
• Exosome precipitation using a rapid method (< 1 hour), requiring only low speed centrifugation
• Optimized sample preparation to minimize carryover of inhibitory compounds in biofluids
• Procedures for rigorous QC of liquid biopsy samples
• Highly sensitive detection system to handle the very low level of RNA found in biofluids
• Highly specific detection method to discriminate between closely related microRNA family members
• Detection method optimized for detection of short microRNA sequences
miRCURY™ Exosome Isolation Kit
Exosome isolation enables detection of
more microRNAs in urine
Vesicles of the correct size are
recovered in the exosome pellet
Exosome isolation enables a larger starting volume of
biofluid to be used, increasing signals.
Nanosight measurements demonstrate that vesicles of
a size range compatible with exosomes are enriched
from urine in the pellet.
2,5
160
2
140
Particles per ml (1010)
microRNAs detected on miRNome panel I
180
120
100
80
60
40
1,5
•Pellet
•Supernatant
1
0,5
20
0
0
0.2
Whole
Urine
0.2
1.5
0
10
200
400
600
800
Size (nm)
Exosomes
Urine starting volume (ml)
Figure 5.
Figure 4.
Rigorous QC of liquid biopsies
qPCR-based QC procedures optimized for biofluids
40
35
Cq value
30
UniSp2 CP
25
UniSp4 CP
20
UniSp5 CP
15
UniSp6 CP
10
5
0
1 3
5 7 9 11 13 15 17 19 21 23 25 27 29 30 33
Urine sample number
Figure 5.
A range of RNA spike-ins are detected by LNA™ qPCR
assays to monitor RNA isolation efficiency, inhibitors,
and detect outlier samples.
miRCURY LNA™ Universal RT microRNA PCR System
High sensitivity and linearity - Ideal for microRNA analysis in liquid biopsies
Urine/CSF
• Sensitive assays are crucial, due to the low RNA
content of biofluids
• Exiqon’s miRCURY LNA™ microRNA PCR assays
are wet-lab validated to have sensitivity and
linearity over a wide range of RNA inputs,
including biofluids
• Red arrow indicates improvement of microRNA
detection from dilute biofluid samples e.g. urine
when using the miRCURY™ Exosome Isolation Kit
Serum/plasma
40
FFPE
35
Tissue, Buffy coat,
PAXgene and cell lines
Cq values
30
25
20
15
10
2pg
20pg
200pg
2ng
20ng
200ng
2µg
Total RNA input in RT reaction
hsa-let-7d-5p
hsa-miR-1
hsa-miR-133a
hsa-miR-145-5p
hsa-miR-194-5p
hsa-miR-451a
Figure 7.
A robust system for accurate microRNA analysis - Validated on biofluids
• In the largest cross-platform comparison study ever (miRQC), Exiqon’s PCR system was the only microRNA
analysis platform to combine both high sensitivity and specificity (Mestdagh et al., Nature Methods 11(8):809-15,
2014)
• We have used the miRCURY LNA™ Universal RT microRNA PCR System to analyze microRNAs in thousands of
biofluid samples including serum, plasma and urine
Step 1: First-strand synthesis (RT)
Mature microRNA
A)
AAAAAAAAAAAAAAAAAAAA
Polyadenylation
B)
AAAAAAAAAAAAAAAAAAAA
TTTTTTTTTTTTTTT
Universal Reverse Transcription:
speed and convenience
5’ universal tag
3’ degenerate anchor
Step 2: Real-time PCR amplification
miR-specific forward primer
A)
LNA
LNA
LNA
TTTTTTTTTTTTTTT
LNA
LNA
LNA
miR-specific reverse primer
B)
Figure 8.
LNA™ in two microRNA specific primers:
sensitivity and specificity
SYBR Green detection:
verification of amplicon
Results
Study overview - microRNA biomarker discovery
DISCOVERY PHASE
Genome wide
screening
Urine sample collection and analysis:
Urine samples collected by hospitals in Denmark
• 3 ml fresh urine (without stabilizer) was centrifuged
to remove cell debris
• Storage in cryotubes at -20 °C (short term) then
-80 °C (long term)
• Exosome and RNA isolation followed by microRNA
qPCR analysis, using the methods shown in Figure 3
Urine samples collected by hospitals in the UK
• Prostate massaged fresh urine (without stabilizers)
was centrifuged to remove cell debris
• RNA isolation without exosome isolation followed by
microRNA qPCR analysis, using the method shown
in Figure 3
VALIDATION PHASE
Discovery screening
on subset
Validation set
miRNA signature
External validation set
miRNA signature
50 individuals
#1: 222 individuals
(22 BPH, 200 PCa)
#2: 227 individuals
(22 BPH, 205 PCa)
#3: 90 individuals
(34 Normal, 56 PCa)
In progress
miRNome PCR Panels
752 microRNAs
Custom PCR Panels
92 microRNAs
Custom PCR Panels
92 or 48 microRNAs
Custom PCR panels
microRNA signature
Identify subset of
relevant microRNAs
Identify candidate
microRNA biomarkers
& endogenous controls
Signature identification
Assess signature
performance
Figure 9.
Genome wide microRNA profiling of cell-free
urine samples:
• Healthy individuals (Benign Prostatic Hyperplasia)
and patients with Prostate Cancer (Stage I-IV)
• A subset of relevant microRNAs were selected for
subsequent discovery screening
microRNA biomarker discovery and validation on
cell-free urine samples:
• Discovery on 222 individuals
• Validation on 227 and 90 individuals from two
independent retrospective cohorts
• Individuals with localized or advanced localized
Prostate Cancer, as well as Healthy Controls
(Benign Prostatic Hyperplasia)
Diagnostic microRNA signatures for prostate cancer in urine
• Differentially regulated microRNAs in urine from prostate cancer individuals were identified (Cohort 1, DK)
• Signatures with diagnostic potential for prostate cancer have been identified using different combinations of
these microRNAs
• Three-microRNA signature: high Area Under the Curve (AUC) was validated in two independent cohorts –
one using sample collection and isolation protocols identical to the discovery cohort (cohort 2, DK) and one
prostate massaged cell free urine without exosome isolation (cohort 3, UK) (Figure 10a)
• The three microRNA signature shows high performance within the intended-use-population (cohort 2 subpopulation, DK) (Figure 10b)
Cohort 2
Cohort 3
1.0
1.0
0.8
0.8
0.8
Sensitivity: 0.88
Specificity: 0.95
Cut-off: >6
0.4
0.2
0.6
Sensitivity
0.6
Sensitivity
Sensitivity
Cohort 1
1.0
Sensitivity: 0.78
Specificity: 0.95
Cut-off: >6
0.4
0.2
0
0.2
0.4
0.6
0.8
1.0
0.4
0
0
0.2
0.4
0.6
0.8
1.0
0
0.2
0.4
0.6
1-Specificity
1-Specificity
1-Specificity
AUC = 0.955
P < 0.001
AUC = 0.892
P < 0.001
AUC = 0.794
P < 0.001
DK – no prostate massage, exosomes isolated
Figure 10a.
Sensitivity: 0.64
Specificity: 0.84
Cut-off: >3
0.2
0
0
0.6
0.8
UK – prostate massaged, no exosome isolation
1.0
Cohort 2 sub-population (PSA< 10 ng/mL)
1.0
Sensitivity
0.8
0.6
Sensitivity: 0.88
Specificity: 0.95
Cut-off: >6
0.4
0.2
0
0
0.2
0.4
0.6
0.8
1.0
1-Specificity
AUC = 0.907
P < 0.001
DK – no prostate massage, exosomes isolated
Figure 10b.
A new diagnostic test (non-invasive) for prostate cancer - intended use:
men with intermediary PSA levels
Today’s practice – based on blood
PSA – indication for Prostate Cancer:
• Not specific for prostate cancer
• 70 % false positives
• 10-20 % false negatives
• Gray-zone: PSA 3-10 ng/mL, 75 % false positives
PSA
< 3 ng/mL
PSA
3 – 10 ng/mL
(gray-zone)
PSA
> 10 ng/mL
New test – based on urine
Negative
Positive
microRNA signature diagnostic for
Prostate Cancer:
• Predict positive biopsy, 90 % accuracy
• Reduce number false positives to < 10 %
• Provide guidance for biopsy
Active surveillance
Figure 11.
TRUS directed biopsy
• Used for tumor grading
• 47 % misdiagnosis
• 30 % false negatives
Conclusions
• Requirements for microRNA analysis in biofluids:
- RT-qPCR system with high sensitivity and specificity
- Rigorous sample QC and standardization
- Enrichment of exosomes is preferred in dilute biofluids
• Exosome isolation enables detection of more microRNAs in dilute biofluids
• The methods developed for sample preparation and LNA™-enhanced microRNA qPCR analysis have been
successfully applied in cell-free urine
• microRNAs in cell free urine are promising non-invasive biomarkers in prostate cancer diagnosis
• A three-microRNA signature has been discovered
• The three-microRNA signature has been validated in independent cohorts
Acknowledgements: Nanosight data kindly provided by iNano (Dr. Ken Howard, Aarhus University).
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