microRNA discovery and biomarker development in clinical samples

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microRNA discovery and biomarker

development in clinical samples

Webinar, February 23rd , 2012, Adam Baker, PhD. [email protected]

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Agenda

• How to study microRNAs – introduction to LNA™

• Detection of microRNAs in clinical samples and

diagnostic development using LNA™ Universal

RT microRNA PCR

• Case Study and Services – Early detection of

colorectal cancer from human patient blood

plasma

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Introduction to microRNAs

Six Facts about microRNAs:

1. Short non-coding RNA molecules of 19-22 nucleotides

2. Post-transcriptional regulators of mRNA

3. 1921 annotated human microRNAs*

4. Regulate at least one third of all human genes

5. Phylogenetically well conserved

6. Altered microRNA expression profiles are associated with a variety of

diseases (cancer, diabetes, neurological disease, viral infection)

*Sanger miRBase release 18.0, November 2011

http://www.mirbase.org


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MicroRNA biogenesis and mode of action

• Perfect complementarity

→ mRNA cleavage

• Imperfect complementarity

→ translational repression and

mRNA decay

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Reasons to use microRNAs as Biomarkers (1)

● Key roles in pathway regulation and tissue differentiation

● Important regulatory roles in many diseases

● Regulate expression of key proteins in drug response pathways

● Predict response to targeted therapies

● Actively secreted into the circulation

● Act as signalling molecules

● Integrate biology from entire organism including diseased tissue

and host response


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Reasons to use microRNAs as Biomarkers (2)

● Highly stable in clinical sample preparations

● Archival FFPE material

● Serum and plasma (routinely collected, minimally invasive)

● Other body fluids

● Relatively small number of genes to profile

● Huge dynamic range (0 to 40,000 molecules per cell)


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Analyzing microRNAs –

challenges using traditional DNA technology

Feature Challenge

Short sequences (19-22 nt) Hard to achieve high sensitivity

Highly homologous families

(single base differences)

Hard to achieve sufficient specificity

Large variation in base composition

(GC content varies between 5-95%)

Hard to design good multiplex assays


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LNA™ technology increases binding affinity

• LNA is a bicyclic high affinity RNA mimic with the

sugar ring locked in the 3’-endo conformation

• Obeys Watson-Crick base-pairing rules

• Stable A-helix with good base-stacking

• Increased Tm (Tm increases by 2 - 8ºC per base)

• Tm normalization (adjust oligos to the same Tm)

• Improved mismatch discrimination

• High sensitivity and specificity in hybridization

assays

K. Bondensgaard et al., Chem. Eur. J. 2000, 6, 2687

M. Petersen et al., J. Am. Chem. Soc. 2002, 124, 5974

LNA™ technology overview


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Analyzing microRNAs –

overcoming challenges by using LNA™

Feature Challenge LNA™ allows

Short sequences (19-22 nt) Hard to achieve high

sensitivity

Increased affinity

Highly homologous families

(single base differences)

Hard to achieve

sufficient specificity

Single nucleotide

discrimination

Large variation in base

composition (GC content

varies between 5-95%)

Hard to design good

multiplex assays

Tm normalization


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Variation in full length DNA capture probe Tm due to variations

in microRNA sequences


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DNA capture probes have decreased sensitivity for low

Tm targets and decreased specificity for high Tm targets


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Optimally designed LNA™ capture probes result in

unmatched sensitivity and specificity


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What are your challenges of microRNA research?

Please reply to the survey, we will display the results at the end of the webinar.


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Agenda




RT microRNA PCR



plasma


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Clinical tissue samples Bio-fluids

Clinical application of the miRCURY LNA™

ISH and Universal RT microRNA PCR System


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LNA™ based high Resolution ISH


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Exiqon has developed a robust ONE DAY microRNA

ISH protocol for manual and automated hybridization

One day

Protocol


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LNA™ ISH technology: miR-21 targets tumor suppressor

& Fibroblast activation


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LNA™ ISH technology: High resolution miR 126 ISH.

Tumor biology of microRNAs in action


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Clinical application of the

miRCURY LNA™ Universal RT microRNA PCR System


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LNA™ Universal RT microRNA PCR System

Advantages:

● Universal RT → no bias,

no pre-amplification

● LNA™ in two specific

primers → sensitivity and

specificity


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microRNA PCR profiling from as little as 0,18mm2 of

FFPE tissue or 20 µl of plasma

● 20 µl plasma/serum or 0,18mm2

tissue / One RT reaction

● 96 or 384-well PCR including

calibrators / controls

● QC / normalization and data

analysis


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Extreme sensitivity allows microRNA PCR profiling

from clinical samples


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Extensive QC and data analysis pipeline

• Automated, user-guided QC

pipeline

• Data flagging and cleanup

customized for qPCR platform

• Diverse normalization protocols

implemented

• Comprehensive QC report

• Visualization of potential

technical and sample biases

• Identify samples that may be

affected by contamination from

blood cells


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Normalization of serum/plasma microRNA qPCR data

is challenging

● Normalization adjusts for technical biases (RNA amount, quality etc)

● No housekeeping genes are stably expressed in all situations

● U6 and 5S are not applicable to serum/plasma samples

● With qPCR panels, no prior assumptions about housekeeping genes

● Select a normalization method appropriate for the dataset

Raw data values Quantile normalization Mean centered

normalization Mean centered

(top 50 expressed)

normalization


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Clinical tissue samples Bio-fluids

Application of the miRCURY LNA™ microRNA

PCR System in biofluids


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Exiqon Diagnostics optimized RNA isolation protocol from serum / plasma (Andreasen et al., Methods 50 (2010) S6–S9)

You have to be able to detect the microRNAs present in

very limited clinical samples


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Blood collection tubes and subsequent

effect on microRNA qPCR

Cq

-va

lue

s


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Plasma or serum

Near perfect linearity at very low concentrations as

found in plasma and serum

● Serial dilution of a

pool of 647 synthetic

microRNAs spiked

into plasma


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Circulating microRNAs are promising

biomarkers

Serum miR-21 associated with relapse-free survival in diffuse large cell B

lymphoma

Lawrie et al., BJH, 2008

human prostate cell line implanted into mice and tumor-derived miRNAs

identified in blood

Mitchell et al., 2008, PNAS,

105(30):10513.

lung, colon cancer and diabetes have specific serum-miRNA profiles Chen et al., 2008, Cell Res, 1-10

microRNAs differentially expressed in serum of ovarian cancer patients Resnick et al., 2009 Gyn

Oncology 112: 55-59

microRNAs differentially expressed in circulating tumor-derived exosomes of

ovarian cancer patients

Taylor et al., 2008, Gyn

Oncology, 110:13

Plasma miR-208 as a Biomarker of Myocardial Injury Ji et al., 2009, Clin. Chem. 55: 11

Circulating microRNAs, potential biomarkers for

drug-induced liver injury

Wang et al., 2009, PNAS


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microRNAs are actively secreted into the

circulation and are stable in serum / plasma

● Three mechanisms of export:

● Exosomes

● RNA binding proteins

● HDL

● Packaging stabilizes the

endogenous microRNAs in

serum/plasma

Cortez, M. A. et al. Nat. Rev. Clin. Oncol. 8, 467–477 (2011)


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microRNAs are actively secreted into the

circulation and are stable in serum / plasma

MicroRNAs in body fluids--the mix of hormones and biomarkers. Cortez et al., 2011, Nat Rev Clin

Oncol, 8(8):467-77.

Export of microRNAs and microRNA-protective protein by mammalian

cells. (nucleophosmin 1, export is energy dependent)

Wang et al., 2010, Nucleic Acids

Res. 38(20):7248-59.

Argonaute2 complexes carry a population of circulating microRNAs

independent of vesicles in human plasma.

Arroyo et al., 2011, PNAS,

108(12):5003-8.

MicroRNAs are transported in plasma and delivered to recipient cells

by high-density lipoproteins.

Vickers et al., 2011, Nat Cell Biol.,

13(4):423-33.

Exosome-mediated transfer of mRNAs and microRNAs is a novel

mechanism of genetic exchange between cells.

Valadi et al., 2007, Nat Cell Biol.,

9(6):654-9.

Glioblastoma microvesicles transport RNA and proteins that promote

tumour growth and provide diagnostic biomarkers.

Skog et al., 2008, Nat Cell Biol.,

10(12):1470-6.

Secretory mechanisms and intercellular transfer of microRNAs in

living cells.

Kosaka et al., 2010, J Biol Chem.,

285(23):17442-52.

Selective release of microRNA species from normal and malignant

mammary epithelial cells.

Pigati et al., 2010, PLoS One,

5(10):e13515.


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microRNAs in serum have been identified to be

potential biomarkers in many diverse diseases


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Dynamic

Window

Of miRNA

Expression

In serum

Normal reference range – Human serum Normal reference range – Human serum 1400+ human samples with over 1 million PCR data points make up the reference

range


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Normal reference range for other biofluids

Human urine Profile

Dynamic

Window

Of miRNA

Expression

In serum


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Which sample types do you work with?

Please reply to the survey, we will display the results at the end of the webinar.


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RT microRNA PCR



plasma


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• Estimated new cases in 2010 in the USA: 142,570

• Estimated deaths in 2010 in the USA: 51,370

• Early diagnosis results in resectable cancer with much improved

prognosis

• BUT around 50% of patients are diagnosed at Stage III / IV

Stage 5 yr relative

survival

Treatment

0-I 93% Surgery

II 80% Surgery/discretionary

adjuvant chemotherapy

III 58% Surgery/adjuvant

chemotherapy

IV 6.9% Chemotherapy

Unmet need for minimally invasive early

detection test of Colorectal Cancer


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Exiqon’s discovery process and PCR platform

are compatible with clinical procedures


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• 50 controls

• 50 CRC patients

• 742 miRNA

screen

• Genome wide

• Multiple QC

check

• Data flagging

• Normalization

• Data analysis

• Quality control

• ROC curve

• miRNA selection

• 76 controls • 151 CRC patients • Focused

Serum/Plasma panel

• Multiple controls

Genome wide

screening

Normalization, QC,

processing

Bioinformatics, data

analysis

Candidate miRNA

discovery screen

• 3000 patients

• Defined miRNA

signature

• Pick & Mix panel

• Multiple controls

Validation Set

miRNA signature

DISCOVERY PHASE VALIDATION PHASE

Development of microRNA Early Detection

Test of CRC in blood plasma


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All Samples Hospital 1 Hospitals 2-5

Sensitivity * 75% 80% 73%

Specificity * 80% 78% 82%

(n) Cancer 151 49 102

(n) Control 76 36 40

All Samples (n=227) Hospital 1 (n=85) Hospitals 2-5 (n=142)

* The same cutoff score was applied on all samples in the study

Focused panel of microRNAs in plasma

may be used as biomarker for CRC


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Development of sensitive quantitative PCR platform

Optimized for clincial serological samples

Conventional PCR. Directly implement in clinical labs

Biomarker pre-discovery

• 50 stage II CRC + 50 matched healthy (colonoscopy-verified)

• Multi-centre, prospective protocol

Biomarker complete discovery

• 151 stage II/III,76 healthy sex age matched (colonoscopy-verified)

• Data Analysis Signature development.

Assay development and retrospective validation

• 3000 symptomatic individuals

• Prospective protocol, multi-centre trial, blood drawn pre-endoscopy

• Complete clinical information (other diseases, medicine usage)

Prospective validation

• 5000 symptomatic individuals

• Prospective protocol, multi-centre trial, blood drawn pre-endoscopy

• Complete clinical information (other diseases, medicine usage)

2009

2010

2011

2012

microRNA Early Detection Biomarker from

Discovery to Development


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Genome wide

screening

Normalization,

QC, processing

Bioinformatics,

data analysis

Candidate miRNA

discovery screen

Validation Set

miRNA signature

DISCOVERY PHASE VALIDATION PHASE

microRNA Biomarker discovery workflow

and panel selection options


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Service Programs at Exiqon

• Customized Assay development, screening, biomarker

discovery or companion diagnostic project development

Customized Sample preparation method development

Custom discovery,development and validation projects

• Full Experimental Design Guidance

Consultation on experimental setup for best data quality

Broad experience in biomarker discovery, development and validation

projects

• GLP processes

Standard operating procedures

Process control and QC checks to demonstrate performance

• Industry Leading Data analysis

Fully customized data analysis package to fit customers needs.

Comprehensive QC processing and statistics.

Webex, teleconference and on site support augment data analysis

and provide industry leading project feedback.


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Pre clinical Animal Models

• Customized sample prep, biomarker isolation, Assay

development, biomarker screening

Customized Sample preparation method devlopment

Toxicology studies, xenograph, dose response studies

• Full animal Experimental Design Guidance

Consultation on specific experimental setup for best data quality

Broad experience in biomarker animal model studies, pre clinical

and IND /NDA applications.

• miRCURY LNA™ Universal RT MicroRNA PCR

Standard operating procedures for animal work

Process control and QC checks to demonstrate performance

• Industry Leading Data analysis

Fully customized data analysis package to fit customers needs.

Comprehensive QC processing and statistics.

Webex, teleconference and on site support augment data analysis

and provide industry leading project feedback.


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● microRNAs are promising biomarkers

● microRNAs can be sensitively and reproducibly detected in clinical

samples.

● Exiqon has developed a one day protocol that allows for sensitive

detection of microRNAs in clinical tissue samples.

● Without pre-amplification, the miRCURY LNA™ Universal RT microRNA

PCR system provides unrivalled sensitivity and specificity in clinical tissue

and biofluid samples.

● Flexible platform enables whole genome profiling and focused /

customizable panels

● A plasma microRNA signature for colorectal cancer was developed and

performs well in samples from independent hospitals

Conclusions


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Acknowledgements

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Webinar, February 23rd , 2012, Adam Baker, PhD. [email protected]

THANK YOU FOR YOUR ATTENTION

microRNA discovery and biomarker development in clinical samples

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