Comparison of Methods for Preserving Morphological, Molecular, … · 2018. 12. 2. · - 23 -...

Comparison of Methods for Preserving Morphological, Molecular, and Protein Biomarkers in Tissue

PAXgene is a trademark of PreAnalytiX GmbH. All other brands are trademarks of their respective owners. © 2012 PreAnalytiX

Lynne Rainen, Ph.D.Scientific DirectorPreAnalytiX GmbHSample Prep and Target Enrichment in Molecular DiagnosticsApril 10, 2013

Presentation Outline

� Current tissue preservation methods

� Introduction to the PAXgene Tissue System

� Comparison of PAXgene Tissue to FFPE and fresh froz en tissue

– Morphology

– RNA, DNA, miRNA

– Proteins

� Acknowledgements

� Questions

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– Morphology

– RNA, DNA, miRNA

– Proteins


� Questions

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Current methods do not simultaneously preserve tiss ue morphology and biomolecules.

Neutral bufferedformalin (NBF)

Liquid nitrogen or RNAlater TM

� Crosslinking of biomolecules

� Lack of standardization

� Morphology destroyed or compromised

MorphologyPreservation:

Nucleic Acid Preservation:

Drawbacks:

Two samples needed for high quality biomolecules and histomorphology.

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Preanalytical variables highly influence sample qua lity.

AnalysisAnalysisAnalytePurificationAnalytePurification

PET StoragePET StorageProcessingProcessingFixationFixationResectionResection

� Ischemia time

� Fixation conditions

� Transport conditions

� Processing/embedding temperatures

� PET storage time/temperature

� Biomolecule isolation protocol





– Morphology

– RNA, DNA, miRNA

– Proteins


� Questions

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- 7 -

The *PAXgene Tissue System consists of a specimen container and companion nucleic acid isolation kits .

PAXgene Tissue ContainerRNA & miRNA

PAX

gene

Tiss

ue K

itsDNA

RNA

PAXgene Tissue FIXPAXgene Tissue STABILIZER

Transport & Store

*For research use only. Not for use in diagnostic p rocedures.

The PAXgene Tissue System simultaneously preserves tissue morphology, DNA, RNA, miRNA and proteins.

AnalysisAnalysisProcessingProcessingStabilization forTransport & StorageFixationResectionResection

H&EPAS

RNA

DNA

IHC

Proteins

ISH

miRNA

2 - 24hours

7 d @ 22°C4 w @ 4°C

*3 y @ -20°C*3 y @ -80°C

-8-*Studies ongoing.

PFPE Tissue





– Morphology

– RNA, DNA, miRNA

– Proteins


� Questions

- 9 -

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Tissue can be fixed in as little as two hours or up to 24 hours in PAXgene with no adverse effect on morpholo gy.

PFPE 3 hours PFPE 24 hours FFPE 24 hours

Thy

roid

M

uscl

e

Liv

er

Kap M. et al., PLoS One. 2011;6(11)

http://www.preanalytix.com/product-catalog/tissue/tissue-atlas/

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Pathologists grade PAXgene fixed tissue equal to or better than formalin-fixed tissue for morphology.

� 26 human tissue types

� Mirrored samples: FFPE and PFPE

� Evaluation performed by four pathologists from three clinical institutions

� Scoring system: -2 to +2 relative to FFPE

� Nuclear details

� Cytoplasmic details

� Membrane details

� Contrast

� Overall impression

“Histological Assessment of PAXgene Tissue Fixation and Stabilization Reagents”

Kap M. et al., PLoS One. 2011;6(11)

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IHC of Human tonsil: Ki-67, clone MIB-1, labeled streptavidin-biotin (LS AB) assay

10 min 70°C, Tris/EDTA buffer pH 9

IHC can be optimized in PAXgene fixed tissue to yiel d results comparable to formalin-fixed tissue.

PF

PE

PF

PE

FF

PE

20 min 98°C, citrate buffer pH 6

PF

PE

PF

PE Optimization

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PFPE tissue is comparable to FFPE tissue in ER α and PR IHC assays.

ERαααα(1D5)

www.preanalytix.com/…/tissue-atlas

PR (1A6)

Case 1 Case 2 Case 3

FFPE PFPE FFPE PFPE

FFPE PFPE FFPE PFPE

FFPE PFPE

FFPE PFPE

IHC of Human Breast cancer: labeled streptavidin- biotin (LSAB) assay

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PFPE tissue is comparable to FFPE tissue in HER2SPoT-Light® CISH assay.

Hu Br Ca:Cases 1 and 2: Her2 +++Cases 3 and 4: Her2 +

CISH Conditions FFPE:- 15 min 98°C- 5 min enzymatic digestion

CISH Conditions PFPE:- no heat pretreatment- no enzymatic digestion

Case 1 Case 2

Case 3 Case 4

FFPE PFPEFFPE PFPE FFPE PFPEFFPE PFPE

FFPE PFPEFFPE PFPE





– Morphology

– RNA, DNA, miRNA

– Proteins


� Questions

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- 16 -

The PAXgene Tissue System maintains tissue morphology while preserving RNA integrity.

Liver SpleenKidney Intestine Lung Brain

RIN 7.3 RIN 6.6 RIN 7.9 RIN 8.4 RIN 7.9 RIN 7.2

4 hours fixation, 7 days stabilization at 22°C

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RNA from the PAXgene Tissue System is free of chemical modifications.

FFPEPFPEFF

FFPE RIN 7.7

PFPE RIN 7.3

Agilent Bioanalyzer Results of RNA from Rat Liver

FF = fresh frozen

PFPE = PAXgene fixed, paraffin-embedded

FFPE = formalin-fixed, paraffin-embedded

Groelz, D., Sobin, L., Branton, P., Compton, C., Wyr ich, R., Rainen, L 2013 Experimental and Molecular Pathology , 94 ( 1 ) pp. 188 - 194 .

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RNA from the PAXgene Tissue System performs betterin RT-PCR than RNA from formalin-fixed tissue.

Groelz, D., Sobin, L., Branton, P., Compton, C., Wyr ich, R., Rainen, L 2013 Experimental and Molecular Pathology , 94 ( 1 ) pp. 188 - 194 .

-4

-2

0

2

4

6

8

10

12

14

16

18

109bp 158bp 189bp 236bp 287bp 331bp 438bp 465bp 569bp 610bp

Del

ta-C

t

FFPE Liver

FFPE Kidney

FFPE Spleen

FFPE Intestine

FFPE LungFFPE Heart

FFPE Brain

FFPE Stomach

PFPE Liver

PFPE Kidney

PFPE Spleen

PFPE Intestine

PFPE LungPFPE Heart

PFPE Brain

PFPE Stomach

-4

-2

0

2

4

6

8

10

12

14

16

18


Del

ta-C

t

FFPE Liver

FFPE Kidney

FFPE Spleen

FFPE Intestine

FFPE LungFFPE Heart

FFPE Brain

FFPE Stomach

PFPE Liver

PFPE Kidney

PFPE Spleen

PFPE Intestine

PFPE LungPFPE Heart

PFPE Brain

PFPE Stomach


* ∆CT = CT (RNA from PFPE or FFPE) – CT(RNA from FF).

*∆C

Tβ-Actin genePrimers

PFPERIN = 4.20

PFPERIN = 4.20

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“A New Technology for Stabilization of Biomolecules in Tissues

for Combined Histological and Molecular Analyses”

RN

A fr

om h

uman

live

r

RIN = 2.20

FFPE

RIN = 2.20

FFPE

RIN = 8.00

Fresh Frozen (FF)RIN = 8.00

Fresh Frozen (FF)

Summary of > 800 RT-qPCR assays

PAXgene fixed tissue RNA compares to RNA from fresh frozen tissue in multiplexed RT-qPCR assays.

Viertler C, et al., J Mol Diagn. 2012 Sep;14(5):458 -66.

FF PFPE 24 hPFPE 3 hFFPE 24 h

GENES (SORTED BY CT FF)

CT

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PFPE tissue is comparable to FF tissue in gene expression *array for molecular mechanisms of cance r.

*LifeTechnologies Human Molecular Mechanisms of Cancer Gene Expression Array

Human Molecular Mechansims of Cancer FFPE vs. Frozen - Normalization to av HKG

R2 = 0,6112

-4

-2

0

2

4

6

8

10

-4 -2 0 2 4 6 8 10

DCT (18s) FFPE

DC

T (

18s)

Fro

zen

Human Molecular Mechansims of Cancer FFPE vs. Frozen - Normalization to av HKG

R2 = 0,6112

-4

-2

0

2

4

6

8

10

-4 -2 0 2 4 6 8 10

DCT (18s) FFPE

DC

T (

18s)

Fro

zen

∆C

t (av

HK

G)

LN2

∆Ct (av HKG) PFPE

∆CT (av HKG) FFPE

Human Molecular Mechanisms of Cancer Human Molecular Mechanisms of Cancer PFPE vs. Frozen - Normalization to av HKGPFPE vs. Frozen - Normalization to av HKG

RR22

-2-2

00

22

44

66

88

1010

1212

1414

1616

-2-2 00 22 44 66 88 1010 1212 1414 1616

DCT PFPEDCT PFPE

DC

T F

roze

nD

CT

Fro

zen

Human Molecular Mechanisms of Cancer PFPE vs. Frozen - Normalization to av HKG

R2

-2

0

2

4

6

8

10

12

14

16

-2 0 2 4 6 8 10 12 14 16

DCT PFPE

DC

T F

roze

n

∆CT (av HKG) PFPE

∆C

T(a

v H

KG

) F

F R2 = 0.963 R2 = 0.611

PFPE vs. FF

∆C

T(a

v H

KG

) F

F

FFPE vs. FF

Combined gene expression array results for three human breast cancer samples.

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The quality and performance of DNA from PAXgene fix ed tissue compares to DNA from fresh frozen tissue.

Hum

an c

olor

ecta

l can

cer

(CR

C)

Human ileum

FF

Viertler C, et al., J Mol Diagn. 2012 Sep;14(5):458 -66.

FF

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miRNA from PAXgene fixed tissue is comparable to miRNA from fresh frozen tissue.

Combined data from three cases of human colorectal cancer

CT FF CT FF

CT

PF

PE

CT

FF

PE

Viertler C, et al., J Mol Diagn. 2012 Sep;14(5):458-66.

R2 = 0.95 R2 = 0.81

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PAXgene tissue can be stored for at least three yea rs at with no adverse effect on morphology or RNA qual ity.

-2

-1

0

1

2

109 bp 189 bp 236 bp 465 bp 569 bp

∆∆ ∆∆C

T

Liver PFPEKidney PFPESpleen PFPEIntestine PFPELung PFPE

-2

-1

0

1

2

109 bp 189 bp 236 bp 465 bp 569 bp

∆∆ ∆∆C

T

Liver PFPEKidney PFPESpleen PFPEIntestine PFPELung PFPE

LiverLiver KidneyKidney SpleenSpleen IntestineIntestine LungLung

Liver 7.4

Kidney 6.7

Spleen 7.5

Intestine 8.1

Lung 6.9 β-Actin RT-qPCR

Avg. RIN

Processing & Paraffin-embedding

Processing & Paraffin-embedding

Storage for 3 years at -80°C

Storage for 3 years at -80°C

Transfer intoPAXgene T.Stabilizer

Transfer intoPAXgene T.Stabilizer

3h Fixationin PAXgeneTissue Fix

3h Fixationin PAXgeneTissue Fix

Resection GrossingResection Grossing

RNA

H&E

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RNA from stored PFPE tissue performs as well as RNA from fresh frozen tissue in real time RT-PCR assays.

* ∆CT = CT (RNA from PFPE or FFPE) – CT(RNA from FF).

RT-qPCR of Rat β-Actin Gene: *∆CT vs. Fresh Frozen (FF) Tissues





– Morphology

– RNA, DNA, miRNA

– Proteins


� Questions

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Proteins and phosphoproteins are preservedin PAXgene fixed tissue.

Ergin et al. J Proteome Res. 2010 Oct 1;9(10):5188-96

Western Blot

Viertler C, et al., J Mol Diagn. 2012 Sep;14(5):458-66.

FF

FF

3 FFPE

1 Frozen

2 PFPE

Phospho-PTEN (Ser380)

Phospho-AKT (Ser473)

Phospho-GSK-3β (Ser9)

Phospho-p38 MAPK (Thr180/Tyr182)

Phospho-HSP 27 (Ser78)

Phospho-p44/42 MAPK (Thr202/Tyr204)

Phospho-NF-κB p65 (Ser536)

Phospho-STAT3 (Ser727)

Phospho-Tyrosin (total)

Phospho-Her2 (Tyr1248)

Phospho-Her3 (Tyr1289)

Phospho-ERα (Ser118)

Phospho-EGFR (Tyr1086)

Phospho-PGR (Ser190)

Phosphorylation-specific antibodies (n=14)

Western blot analysis of phosphoproteins from PFPE samples: study design

Tissue types (n=20)

Ovary Ovarian cancer

Breast Breast cancer

Prostate Prostate cancer

Salivary gland Bladder

Oesophagus Gall bladder

Colon Duodenum

Muscle Tongue

Stomach Thyroid gland

Kidney Lung

Uterus Pancreas

20 different

tissue types

1 2 3

Gündisch et al. PLoS One 2013, in press


The phosphoproteome is quantitatively preserved in PFPE samples.

PAXgene fixation allows for the application of MALD I-IMS.

Ergin et al., J Proteome Res. 2010 Oct 1;9(10)

cryoPFPE

Pancreas

• In contrast to FFPE, PFPE and cryo-preserved pancreatic samples display a multitude of peaks

• Visualization of Insulin (m/z 3485) and Glucagon (m/z 5808) expression in pancreatic tissue.

• Spectra show the differential expression of Insulin and Glucagon comparing exocrine pancreas and endocrine pancreas

PFPE tissue is comparable to cryopreserved tissue i n 2D-PAGE.

Colon

Duodenum


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Summary of PAXgene tissue vs. FFPE and frozen tissue

The PAXgene Tissue System:� Simultaneously preserves histomorphology and biomol ecules.

� Formalin preserves histomorphology, but not biomole cules.� Freezing preserves biomolecules, but not morphology .

� Works without crosslinking or chemical modification .� Formalin crosslinks biomolecules.

� Allows for storage of tissue either in stabilizatio n reagent or as paraffin blocks.

� Gives histology results comparable to FFPE while pr eserving biomolecules comparably to fresh freezing in liquid nitrogen.

PAXgene Tissue enables combined histological and multimodal analysis of biomolecules from the same sample.



� Introduction to the *PAXgene Tissue System


– Morphology

– RNA, DNA, miRNA

– Proteins


� Questions

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PAXgene performance has been proven in more than 10,000 human tissue samples.

Standardization and Improvement of Generic Pre-analytical Tools and Procedures for In Vitro Diagnostics

� European Commission grant EC FP7-HEALTH-2007-B

� Collaborative Research Project: 16 commercial and academic members

� Mission: To provide pan-European quality assurance schemes, guidelines and tools to minimize the effects of pre-analytical conditions on in vitro diagnostic results.

www.spidia .eu

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PAXgene has been chosen as the only fixative for th e NIH/NCI GTEx program.

� The Genotype Tissue Expression Project (GTEx) is an NIH common fund initiative.

� Aims to establish a resource database and tissue bank in which to study the relationship between genetic variation and gene expression.

� Over 170 donors (>5000 tissue samples) have been collected to date.

”PAXgene is at least as good as formalin for histology. It is now our only

morphologic fixative.” OBBR pathologists Philip Branton, John Madden James Robb, Leslie Sobin, Carolyn Compton (Critical Path Institute)

http://www.genome.gov/Pages/About/NACHGR/September2011AgendaDocs/NACHGR_Sep122011_%20GTExUpdate_Struewing.pdf

http://biospecimens.cancer.gov/resources/sops

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Acknowledgments

*QIAGEN GmbH• U. Oelmüller• C. Lenz• N. Dettmann• I. Blassnig• E. Traenert

*PreAnalytiX GmbH• R. Wyrich• E. Provencher• F. Augello

*Erasmus Medical Center Rotterdam (EMC) • P. Riegman• M. Kap• B. de Jong

* Technical University Munich (TUM)• K.-F. Becker• B. Reischauer-Ergin• S. Gündisch

* Medical University of Graz (MUG)• K. Zatloukal• C. Viertler

* Funding for PAXgene Tissue work from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement n°222916.

§ National Cancer Institute• Leslie Sobin • Philip Branton• Kristin Ardlie (Broad Institute) • Carolyn Compton (Critical Path Institute)

§ Funding for PAXgene Tissue work from NCI Contract No. HHSN261200800001E.



� Introduction to the *PAXgene Tissue System


– Morphology

– RNA, DNA, miRNA

– Proteins


� Questions

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Comparison of Methods for Preserving Morphological, Molecular, … · 2018. 12. 2. · - 23 -...

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