A New Technology for Simultaneous Preservation

of Biomolecules and Morphology in Tissues

Facilitates Biomarker Development

BackgroundMolecular characterization of human diseaserequires analysis of multiple parametersranging from histopathology to a broadspectrum of molecular biomarkers. Themorphological characterization is based on theanalysis of formaldehyde-fixed and paraffin-embedded (FFPE) tissues but it is known thatformalin fixation impairs molecular analyseswhich typically require frozen tissue samples.In the context of personalized medicine,upcoming molecular diagnostics and omics-technologies, there is an increasing need forcombined morphological and molecularanalyses from the same tissue sample. Withinthe European FP7 project SPIDIA wedeveloped and tested a new technology(PAXgene Tissue) for high-quality preservationof biomolecules and morphology in paraffin-embedded tissue samples.

MethodsCorresponding samples from different human(non-) malignant tissues were either fixed inbuffered formaldehyde or with PAXgeneTissue and paraffin-embedded (PFPE), snap-frozen tissue samples served as reference. Ina comparative study we investigated thepreservation of morphology, antigenicity,nucleic acids and (phospho-) proteins.

� Preservation of morphology andantigenicity resembling FFPE samples.� Excellent RNA quality and strongcorrelation of multiple mRNA profiles withsnap frozen samples in qPCR andmicroarray analysis.� High molecular mass DNA, well-suited forlong-range and multiplex PCR, and differentsequencing techniques.� Comparable preservation of (phospho-)proteins with snap- frozen samples.

Conclusions

Viertler C 1,9, Groelz D 2,9, Kashofer K 1, Gündisch S 3,9, Reischauer B 3,9,Riegman P 4,9, Winther R 5,9, Wyrich R 6,9, Kruhoffer M 7,9, Walch A 8, Becker KF 3,9, Oelmüller U 6,9, Zatloukal K 1,9

¹Institute of Pathology, Medical University of Graz, Austria, ²PreAnalytiX GmbH, Hombrechtikon, Switzerland, ³Institute of Pathology, Technical University of Munich, Germany, 4Department of Pathology, Erasmus MC, Rotterdam, The Netherlands,5Dako Denmark A/S, Glostrup, Denmark, 6QIAGEN GmbH, Hilden, Germany, 7AROS Applied Biotechnology A/S, Aarhus, Denmark, 8Institute of Pathology, Helmholtz Center Munich, Neuherberg, Germany,

9The SPIDIA consortium, www.spidia.eu

PFPE samples show well-preserved morphology andantigenicity, comparable to FFPE samples. For someantibodies less harsh retrieval procedures can be used.(a) H&E staining of human stomach tissue. (b) CD 20 staining of humanspleen tissue (c) K8/18 staining of human liver.

Western blot analysis of phosphoproteins in PFPEhuman tissue samples : visible protein bands ofPFPE extracts are comparable to snap frozensamples and the phosphorylation levels are well-preserved.

Results

Morphology and Antigenicity

0

2

4

6

8

10

P1T1

P2T3

P2T0

P1T0

P2T2

P2T1

P5T2

P5T0

P4T2

P1T3

P4T1

P5T1

P1T2

P4T0

P3T1

P3T0

P3T2

P5T3

P3T3

RIN

Score

Patient number (P) and Time point (T)

Cryo

PAX

1.9

1.95

2

2.05

2.1

2.15P1T0

P1T1

P1T2

P1T3

P2T0

P2T1

P2T2

P2T3

P3T0

P3T1

P3T2

P3T3

P4T0

P4T1

P4T2

P5T0

P5T1

P5T2

P5T3

Purity

260/280

Patient number (P) and Time point (T)

Cryo

PAX

(b)

DNA integrity& performance in multiplex PCR of PFPE samples .(a) High molecular mass bands indicating good DNA integritywere visible for PFPE and cryo-preserved samples but not forFFPE samples. Genomic DNA extracted from corresponding FFPE, PFPE, andsnap-frozen (CRYO) samples of 5 human breast cancer cases was separated on1% agarose gels and visualized with ethidium bromide.(b) Multiplex PCR of eight fragments of different human gene s.PRNP (222 bp), CD79b (310 bp), cKIT (414 bp), AGTR2 (523 bp), CD14 (662bp), CD40 (756bp), CD59 (845 bp) and CD19 (955 bp).

(a)

(b)

Proteomic Applications

References: Viertler et al. J Mol Diagn. 2012 Sep;14(5):458-66. Epub 2012 Jun 28. Ergin et al. J Proteome Res. 2010 Oct 1; 9(10):5188-5196.

Contact& further information: christian.viertler@medunigraz.at; www.spidia.eu

Nucleic Acids

High correlation of gene signature in PFPE and cryo-preserved samples (R²=0,99), but decreased correlation(R²=0,89) and major gene-to-gene variations in FFPEsamples.Gene expression analysis of corresponding human liver samples byqRT-PCR on predefined TaqMan array “Human Molecular Mechanismsof Cancer” plate. Delta Cts were calculated using the frozen sample asreference.

New Tissue Stabilisation Technology

P A X g e n e T i s s u e p r o v i d e s

� Simultaneous high-quality preservation ofbiomolecules and morphology in clinicaltissue samples.� Direct correlation of morphologicaldisease phenotypes with alterations ofbiomolecules.� Multimodal biomarker studies in a routineclinical setting.� Molecular analyses of lesions where acollection of snap-frozen material isimpossible.

I nnovat ive Aspe c ts

High-throughputexpressionprofiling revealshigh correlationbetween PAXgene-fixed and snapfrozen samples.GDM analysis showingthe Euclidian distancebetween samples basedon expression valuesfrom all genes. Thevariable that contributesmost to the distancebetween samples is theindividual and notischemia time or samplestabilization method.

Impact of ischemia time and stabilization method analysed wi thAffymetix Human Genome U219 Array on the GeneTitan hybridization,wash and scanning station. Liver needle biopsies from 5 patients (P1-5), exposed todifferent ischemia time points (T0-3), were either snap-frozen (Cryo) or fixed andstabilised with PAXgene Tissue (PAX). (a) RNA quality control (b) GDM analysis.

The work leading to these results has received funding from the EUSeventh Framework Programme under grant agreement n°222916.

MALDI imagingmass spectrometry.In contrast to FFPE, PFPEand cryopreservedpancreatic samples displaya multitude of peaks(upper panel).Visualization of Insulin andGlucagon expression(lower panel: left/PFPE,right/cryopreservedsamples).

PFPE samples arecomparable to cryo-preserved samplesin reverse-phaseprotein microarray(RPPA) experiments.

Quality control using Agilent Bioanalyzer shows comparableRNA integrity (RIN score) and purity (OD 260/280 ratio) of PF PEand cryopreserved samples.

High correlation ofmicroRNAexpression inPFPE and snap-frozen samples(R²= 0.95), whereasFFPE samplesshowed a lowercorrelation (R²=0.81). MicroRNAs fromcorresponding aliquotsof three colon cancercases were quantifiedby real-time RT-PCR ona TaqMan 7700.

(a)