technology seminar, april 6 , 2016 sample to insight...

Sample to Insight

Technology Seminar, April 6th, 2016 Sample to Insight: Integrated Solutions for Liquid Biopsy – Rapid Isolation of Exosomes, Other Extracellular Vesicles and Vesicular RNA

Martin Schlumpberger

Associate Director Scient. Appl. - Sample Technologies

University of Hong Kong – Technology Seminar, April 6th, 2016

Sample to Insight

Liquid Biopsy - Innovation is in the blood

A minimal invasive technology for detecting signs of cancer and other diseases

Liquid Biopsy has the potential to transform healthcare


Sample to Insight

Liquid Biopsy - The most cited analyte domains

Which approach to choose?

Free circulating DNA

Circulating Cell-Free Nucleic

Acids (ccf-NA)

Low amount of DNA

Not all tumors shed DNA

DNA highly fragmented

Background of WT/maternal

DNA

CTCs

Circulating Tumor and containing nucleic acids

Very low abundance: 1–10

CTC/ml blood

Isolation procedure

EMT (Epithelial -

Mesenchymal Transition)

Mostly in metastatic stage

High background of normal

cells

Exosomes

and extracellular vesicles containing DNA, RNA & proteins

1010-12 exosomes/ml plasma

Lengthy and non specific

isolation procedures

Smaller than CTCs and

lower number of copies per

biomarker

Clinical utility in evaluation


Sample to Insight

Liquid Biopsy

4

Complete and modular workflow coverage

ccfNA

QIAamp ccfNA

QIAsymphony DSP

AXpH DNA Kit

PCR portfolio (incl.therascreen)

QIASeq / GneRead Library Prep (NGS)

Gene Read Targeted Panels

Bioinformatics

Solutions

CTC

AdnaSelect Repli-g (single cell) WGA / WTA

AdnaDetect

PCR portfolio

AdnaDetect

Bioinformatics

Solutions

AdnaSelect REPLI-g single cell DNA Library Prep Kit

REPLI-g single cell RNA Library Prep Kit

Bioinformatics

Solutions

Exo-

somes

exoEasy exoRNeasy miScript PreAMP

RT² PreAMP

miScript (PCR)

RT2 Profiler (PCR)

Bioinformatics

Solutions

exoEasy REPLI-g single cell RNA Library Prep Kit (NGS) Bioinformatics

Solutions

Sample

collection

& Analyte

enrichment

Sample

Isolation Amplification Detection

Data

Analysis &

Interpretation


Sample to Insight

Frequency of CTC in whole blood of progressed cancer patients

As low as 1-10 CTCs/ml blood

Background of millions of normal cells

Transcription of tumor associated mRNA by normal nucleated blood cells

Epithelial to Mesenchmyal Cell Transition (EMT)

Molecular characterization of circulating tumor cells - Challenges

Circulating Tumor Cells – a cornerstone for understanding caner

Selective enrichment of tumor cells or removal of other nucleated cells in blood

Sample to Insight

The CTC AdnaTest

CTC enrichment platform for the detection of circulating tumor cells in blood

Direct detection of disseminated tumor cells in patient's blood by analysis of the

tumor associated gene expression

Within treatment monitoring, a conclusion concerning successful cancer therapy

(surgery, chemotherapy, radiation) may be reached early on

Detection of tumor cells in the circulation may indicate recidives in the course of

cancer aftercare

Sample to Insight

The CTC AdnaTest

CTC AdnaTest

The AdnaTest is the unique combination

of AdnaSelect for the isolation of CTCs

and AdnaDetect for the detection of

tumor associated marker

Product range:

AdnaTest ColonCancer

AdnaTest BreastCancer

AdnaTest ProstateCancer

AdnaTest OvarianCancer

AdnaTest EMT/StemCell

Sample to Insight

How does the AdnaTest work?

Optimized combination of antibodies for tumor cell selection and the

combination of tumor-associated markers for the detection of tumor cells e.g.:

EpCAM,

CA15-3,

Her2

….

AdnaTest Technology

COCP

Proprietary “Combination-of-

Combinations-Principle”

Sample to Insight

The CTC AdnaTest is a 2-step process

Detection of tumor cells in peripheral blood

Immunomagnetic cell selection with multiple tumor-associated

antibodies

AdnaTest Select AdnaTest Detect

Subsequent molecular characterization

RT-PCR analysis

Two component model to allow blood & CTC collection and analysis at different places

Sample to Insight

The CTC AdnaTest – complete workflow

Sample to Insight

AdnaTests available

AdnaTest Target Genes

AdnaTest BreastCancer (CE) Muc-1, Her2, EpCAM, ER/PR

AdnaTest ProstateCancer (CE) PSA, PSMa, EGFR, AR

AdnaTest ColonCancer (CE) EpCAM, EGFR, CEA

AdnaTest OvarianCancer (CE) EpCAM, Muc-1, CA-125, ERCC1

AdnaTest OvarianCancer-2 (non-CE) EpCAM, Muc-1, CA-125, ERCC1

AdnaTest EMT-1/StemCell (non-CE) Muc-1, Her2, EpCAM, ALDH1, Pi3K, Akt2,

Twist

AdnaTest EMT-2/StemCell (non-CE) ALDH1, Pi3K, Akt2, Twist + choice of

Breast, Colon, Prostate or ovarian PCR

Sample to Insight

Liquid Biopsy - The most cited analyte domains

Which approach to choose?

Free circulating DNA

Circulating Cell-Free Nucleic

Acids (ccf-NA)

Low amount of DNA

Not all tumors shed DNA

DNA highly fragmented

Background of WT/maternal

DNA

CTCs

Circulating Tumor and containing nucleic acids

Very low abundance: 1–10

CTC/ml blood

Isolation procedure

EMT (Epithelial -

Mesenchymal Transition)

Mostly in metastatic stage

High background of normal

cells

Exosomes

and extracellular vesicles containing DNA, RNA & proteins

1010-12 exosomes/ml plasma

Lengthy and non specific

isolation procedures

Smaller than CTCs and

lower number of copies per

biomarker

Clinical utility in evaluation


Sample to Insight

Scanning EM of exosomes shedding into the

blood from a GBM brain cancer cell –

published in Nature Cell Biology , Dec 2008,

Skog et al.

MVs/exosomes shedding from cells

What are exosomes?

Transmission EM of Multi Vesicular

Bodies in Kidney, courtesy of Leileata

Russo


Sample to Insight

Exosomes/Microvesicles (MVs)


It’s the cargo that matters

MVs/exosomes are ca. 50-

200 nm small vesicles

excreted by all cells

MVs/exosomes are found in

all biofluids (e.g. blood)

MVs/exosomes contain stable

nucleic acids (mRNA,

microRNA, other small

RNAs), DNA, and protein,

protected from degradation

by a lipid bilayer

Contents are specifically

packaged, mechanism of

local and distant cellular

communication

Cell

MVs

Exosomes

Blood Plasma

Sample to Insight

Integrity of EVs isolated by exoEasy


EVs were isolated from 4 ml of plasma (collected by apheresis) using the exoEasy Maxi Kit

Visualization by cryo-EM imaging on perforated carbon film (dark grey areas)

Fully intact, spherical vesicles of different sizes between approximately 50-400 nm diameter

Some vesicles distorted or clustered

Similar to EVs prepared by ultracentrifugation

Sample to Insight

Uptake of Isolated EVs into target Cells


Dye only (neg. control) Dye + eluted EVs

Bodipy

(vesicles)

DAPI

(Nuclei)

Vesicles produced in HEK 293T cells, isolated using exoEasy, buffer change by UF, labeled using

Bodipy-ceramide, excess dye removed using SEC

For uptake in 293T cells: approx. 1% of total eluate (1.45E9 particles acc. to Nanosight) applied to

2x104 cells for 1h

Good uptake, vesicles are internalized by endocytosis (in this case), i.e. no fusion with plasma membrane

Sample to Insight

Uptake of Isolated EVs into Primary Cells


Vesicles produced in HEK 293T cells, isolated using exoEasy, buffer change by UF, labeled using

Bodipy-ceramide, excess dye removed using SEC

For uptake: approx. 1x10e4 labeled particles per cell (quantitated by Nanosight) applied to cells for 1h

Good uptake, vesicles are internalized by endocytosis (in this case), i.e. no fusion with plasma membrane

Dye only (neg. control) Dye + eluted EVs

Bodipy

(vesicles)

DAPI

(Nuclei)

Merge

Sample to Insight

exoRNeasy Serum/Plasma Kits

Flowchart


Sample to Insight

Binding capacity of exoEasy Maxi column


0.5ml

Plasma

1.0ml

Plasma

2.0ml

Plasma

4.0ml

Plasma

8.0ml

Plasma

16ml

Plasma

linear increase of signal

Sample to Insight

,

Both methods purify RNA of similar size and yield

exoRNeasy isolates small and large RNAs from EV

Bioanalyzer sizing

2 ml plasma was

pre-filtered (0.8

µm) to exclude

larger particles.


Sample to Insight

EVs contain both, small and large RNAs

Purification of large, intact, non-degraded RNAs from EVs


exoRNeasy total RNA

small RNA fraction large RNA fraction

Sample to Insight

EV contain full length mRNAs with intact poly A tails

mRNA transcripts are not degraded


3 kb from polyA 5 kb from polyA

1 kb from polyA 1 kb from polyA 1 kb from polyA BRAF

EGFR

GAPDH HPRT1

KRAS

Sample to Insight

* Arroyo, J.D. et al. (2011) Argonaute2 complexes carry a population of circulating microRNAs

independent of vesicles in human plasma. Proc. Natl. Acad. Sci. USA 108, 5003.

exoRNeasy captures all mRNA & vesicle-specific miRNAs

mRNA exclusively within vesicles – near 100% bound

miRNA in vesicular and non-vesicular fractions

(e.g. free Ago2 complexes)


Sample to Insight

Comparison of miRNA populations in plasma

Abundance of miRNAs inside and outside of extracellular vesicles


Mainly

inside

vesicles

Mainly

outside

vesicles

Sample to Insight


Analysis of extracellular miRNA in CRC patients plasma

vesic

ula

r n

on

-vesic

ula

r

* - master thesis C. Keup, 2016

Plasma from 6 cancer

patients, 3 healthy controls

Vesicular RNA isolated using

exoRNeasy, non-vesicular

RNA from column flow-through

miRNA analyzed using

miScript serum & plasma PCR

array

Off-the-shelf array allows

distinction of cancer patients

from healthy controls

Distinction possible by both,

vesicular and non-vesicular,

fractions of cell-free miRNA

Sample to Insight


Analysis of extracellular miRNA in CRC patients plasma

* - master thesis C. Keup, 2016

Plasma from 6 cancer

patients, 3 healthy controls

Vesicular RNA isolated using

exoRNeasy, non-vesicular

RNA from column flow-

through

miRNA analyzed using

miScript serum & plasma

PCR array

Individual miRNAs show

strong differences in

abundance between patients

and controls

Sample to Insight

Detection of low-abundance RNAs


exoRNeasy can be scaled up to 4 ml of plasma

CT Value (90 mRNAs, pre-amplified)

Inp

ut

Vo

lum

e

Not detected

46%

mRNAs from known oncogenes are readily detected in

high volumes of plasma

Sample to Insight

Isolated exRNAs are suitable for NGS applications

Detection of somatic mutations from CRC in exRNAs


No

. of

Rea

ds

Freq

uen

cy

wildtype KRAS

25000 100%

20%

No. of reads single variants

10% KRAS G12D WT

MUT 5000

Patient with KRAS G12D positive colorectal cancer (CRC)

2 ml pre-filtered plasma

exRNA isolation using the exoRNeasy Serum/Plasma Maxi Kit

Targeted re-sequencing on an Illumina MiSeq

Over 10% of all reads that matched to the KRAS gene carry the c.35 G>A /

p.G12D mutation previously identified in the primary tumor

Sample to Insight

Exosomal RNA extraction using exoRNeasy


Conclusions

exoEasy allows rapid isolation of intact EVs for

functional studies or subsequent isolation and analysis

of vesicular content

exoRNeasy extracts high quality RNA from plasma EVs

using a fast and convenient spin-column procedure

EVs contain small and large, non-degraded mRNAs

EVs contain plasma mRNA and a specific fraction of

miRNA

EVs carry a small, defined fraction of all plasma miRNAs

which may allow for the detection of specific biological

signals and potentially diagnosis of disease

EVs enable detection of somatic mutations and

oncogene expression

Sample to Insight

Exosomes and Liquid Biopsies Asia 2016, April 7-8th, 2016

QIAGEN

Karolin Spitzer

Corinna Keup

Markus Sprenger-Haussels

Exosome Diagnostics

Christine Coticchia

Daniel Enderle

Mikkel Noerholm

Johan Skog

Enderle, D., et al. Characterization of RNA from Exosomes and Other

Extracellular Vesicles Isolated by a Novel Spin Column-Based Method.

PLoS One. 2015 Aug 28;10(8):e0136133

Acknowledgments

Massachusetts General Hospital

Casey Maguire

Sybren L.N. Maas

Valentina Zappulli

Dakai Mu

UMR-CBMN-University of Bordeaux

Alain Brisson

Center for Pathology Kempten-Allgäu

Barbara Dockhorn-Dworniczak

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0136133



Sample to Insight

Other Topics


Exosomes and exosomal RNA from cell culture

supernatants

Stability of EVs through freeze / thaw cycles

Influence of sample handling on results

Sample to Insight

Vesicular RNA from Cell Culture Medium


2 ml HeLa culture medium per prep; exoRNeasy started from fresh cultured

supernatant, or after 1 or 5 freeze/thaw cycles; negative control sample used 0.5% SDS

to lyse vesicles

no difference in RNA recovery even after 5 freeze/thaw cycles

Effect of Freeze / Thaw Cycles on Vesicle Integrity (RNA Yield)

Sample to Insight

Influence of Blood Collection Device on RNA


Sample to Insight

Different Pre-Filtration vs. Centrifugation Conditions


Effect on isolated RNA Effect on eluted vesicles

without filtration or centrifugation, >95% of RNA is from particles >800nm – residual

cells and cell fragments

3000 x g and 0.8 µm filtration remove roughly the same proportion of RNA

Smaller filter pore size remove progressively more RNA; effect stronger for long RNA

transcripts

only 100 nm filter shows noticeable effect on particle diameter and concentration

Sample to Insight

Other Topics


Exosomes and exosomal RNA from cell culture

supernatants

Stability of EVs through freeze / thaw cycles

Influence of sample handling on results

Compatible with wide range of collection tubes, but not

advisable to use different tubes within one study

delay in processing of blood can strongly affect

outcome

Additional centrifugation or filtration is essential to

remove cellular RNA background

Treatment affects representation of different classes of

vesicles and RNA

RNA is not evenly distributed in vesicles

Conclusions

technology seminar, april 6 , 2016 sample to insight...

Documents