A New Technology for Simultaneous Preservation of ... · A New Technology for Simultaneous...
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A New Technology for Simultaneous Preservation of Biomolecules and Morphology in Tissues Facilitates Biomarker Development Background Molecular characterization of human disease requires analysis of multiple parameters ranging from histopathology to a broad spectrum of molecular biomarkers. The morphological characterization is based on the analysis of formaldehyde-fixed and paraffin- embedded (FFPE) tissues but it is known that formalin fixation impairs molecular analyses which typically require frozen tissue samples. In the context of personalized medicine, upcoming molecular diagnostics and omics- technologies, there is an increasing need for combined morphological and molecular analyses from the same tissue sample. Within the European FP7 project SPIDIA we developed and tested a new technology (PAXgene Tissue) for high-quality preservation of biomolecules and morphology in paraffin- embedded tissue samples. Methods Corresponding samples from different human (non-) malignant tissues were either fixed in buffered formaldehyde or with PAXgene Tissue and paraffin-embedded (PFPE), snap- frozen tissue samples served as reference. In a comparative study we investigated the preservation of morphology, antigenicity, nucleic acids and (phospho-) proteins. Preservation of morphology and antigenicity resembling FFPE samples. Excellent RNA quality and strong correlation of multiple mRNA profiles with snap frozen samples in qPCR and microarray analysis. High molecular mass DNA, well-suited for long-range and multiplex PCR, and different sequencing 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, 4 Department of Pathology, Erasmus MC, Rotterdam, The Netherlands, 5 Dako Denmark A/S, Glostrup, Denmark, 6 QIAGEN GmbH, Hilden, Germany, 7 AROS Applied Biotechnology A/S, Aarhus, Denmark, 8 Institute of Pathology, Helmholtz Center Munich, Neuherberg, Germany, 9 The SPIDIA consortium, www.spidia.eu PFPE samples show well-preserved morphology and antigenicity, comparable to FFPE samples. For some antibodies less harsh retrieval procedures can be used. (a) H&E staining of human stomach tissue. (b) CD 20 staining of human spleen tissue (c) K8/18 staining of human liver. Western blot analysis of phosphoproteins in PFPE human tissue samples: visible protein bands of PFPE extracts are comparable to snap frozen samples 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 2.15 P1T0 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 integrity were visible for PFPE and cryo-preserved samples but not for FFPE samples. Genomic DNA extracted from corresponding FFPE, PFPE, and snap-frozen (CRYO) samples of 5 human breast cancer cases was separated on 1% agarose gels and visualized with ethidium bromide. (b) Multiplex PCR of eight fragments of different human genes. PRNP (222 bp), CD79b (310 bp), cKIT (414 bp), AGTR2 (523 bp), CD14 (662bp), CD40 (756 bp), 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: [email protected]; 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 FFPE samples. Gene expression analysis of corresponding human liver samples by qRT-PCR on predefined TaqMan array “Human Molecular Mechanisms of Cancer” plate. Delta Cts were calculated using the frozen sample as reference. New Tissue Stabilisation Technology PAXgene Tissue provides Simultaneous high-quality preservation of biomolecules and morphology in clinical tissue samples. Direct correlation of morphological disease phenotypes with alterations of biomolecules. Multimodal biomarker studies in a routine clinical setting. Molecular analyses of lesions where a collection of snap-frozen material is impossible. Innovative Aspects High-throughput expression profiling reveals high correlation between PAXgene- fixed and snap frozen samples. GDM analysis showing the Euclidian distance between samples based on expression values from all genes. The variable that contributes most to the distance between samples is the individual and not ischemia time or sample stabilization method. Impact of ischemia time and stabilization method analysed with Affymetix Human Genome U219 Array on the GeneTitan hybridization, wash and scanning station. Liver needle biopsies from 5 patients (P1-5), exposed to different ischemia time points (T0-3), were either snap-frozen (Cryo) or fixed and stabilised with PAXgene Tissue (PAX). (a) RNA quality control (b) GDM analysis. The work leading to these results has received funding from the EU Seventh Framework Programme under grant agreement n°222916. MALDI imaging mass spectrometry. In contrast to FFPE, PFPE and cryopreserved pancreatic samples display a multitude of peaks (upper panel). Visualization of Insulin and Glucagon expression (lower panel: left/PFPE, right/cryopreserved samples). PFPE samples are comparable to cryo- preserved samples in reverse-phase protein microarray (RPPA) experiments. Quality control using Agilent Bioanalyzer shows comparable RNA integrity (RIN score) and purity (OD 260/280 ratio) of PFPE and cryopreserved samples. High correlation of microRNA expression in PFPE and snap- frozen samples (R²= 0.95), whereas FFPE samples showed a lower correlation (R²= 0.81). MicroRNAs from corresponding aliquots of three colon cancer cases were quantified by real-time RT-PCR on a TaqMan 7700. (a)
Transcript of A New Technology for Simultaneous Preservation of ... · A New Technology for Simultaneous...
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: [email protected]; 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)
