Transcript Retrospective Survival Studies
BIOBANKING AND MOLECULAR PATHOBIOLOGY WG
MOLECULAR PATHOLOGY WG
Le Biobanche di Campioni Inclusi
Bari, 16/12/14 Giorgio Stanta, Dipartimento di Scienze Mediche Università di Trieste
ARCHIVE TISSUES: IMPROVING MOLECULAR MEDICINE RESEARCH AND CLINICAL PRACTICE
www.impactsnetwork.eu
G. Stanta
BIOBANKING AND MOLECULAR PATHOBIOLOGY WG
MOLECULAR PATHOLOGY WG
Technical working groups # DNA and RNA extraction SOPs and IQC rules in AT (OECI, ESP)- G. Stanta, A. Jung, S. Bonin # NGS in diagnostics and clinical research (ESP)- G. Hoefler, K. Kashofer # Proteomics SOPs in AT (ESP, OECI)- K. Becker # Preanalytical conditions in tissues (ESP, OECI)- M. Dietel, G. Bussolati, A. Sapino # Heterogeneity (inter-WGs of ESP - OECI) – ESP WGs chairman, G. Stanta # CEN–New ISO 15189 rules-DNA, RNA and proteins from tissues – K. Becker, P. Riegman, G. Stanta, K. Zatloukal Courses
BBMRI-ERIC CLINICAL BIOBANKS MEETING
Berlin 17/09/14 ARCHIVE TISSUES WG CLINICAL BIOBANKING WG
G. Stanta
MEDICO-BIOLOGICAL RESEARCH
BASIC RESEARCH ……………… TRANSLATIONAL RESEARCH > 10 years on average > devoted to future patients RETROSPECTIVE CLINICAL RESEARCH short time today’s patients -verification of clinical cases -efficacy of the new therapies -therapy response subgroups -intrinsic and acquired resistance BM -very long follow-up studies -performance evaluation -to establish costs and benefits Applied medicine and clinical research are today an integrated and indissoluble process
G. Stanta
CONCLUSIONS IN REVIEWS AND METANALYSIS……..
“ In conclusion, it is clearly evident from all these studies that, as with previous studies on gene profiling, most emerging miRNA signatures are not fully overlapping. These results might be explained by different specimens (frozen vs paraffin-embedded, micro- vs non-microdissected), experimental platforms used (quantitative PCR vs different miRNA array or in situ hybridization systems), tumour types, stage, and regimens as well as small sample size, ethnic differences in the study populations, lack of multivariate analysis and correction for multiple testing.
”
E Giovannetti et al Critical Reviews in Oncology/Hematology 81 (2012) 103–122
Gene expression signatures for predicting the outcome in stage II colorectal cancer meta-analysis
Takashi Akiyoshi et al “Recent approaches to identifying biomarkers for high-risk stage II colon cancer” Surg Today DOI 10.1007/s00595-012-0324-4 - 2012
Participating Organizations Organization Site Austrian BBMRI Node
www.bbmri.at
BBMRI-ERIC – Biobanking and Biomolecular Resources Research Infrastructure European Research Infrastructure Consortium Czech BBMRI Node
www.bbmri-eric.eu
www.recamo.cz/en/bbmri
EATRIS – European Infrastructure for Translational Medicine ECCO – European CanCer Organisation ECPC – European Cancer Patient Coalition EPAAC – European Partnership for Action Against Cancer
www.eatris.eu
www.ecco-org.eu
www.ecpc.eu
www.epaac.eu
ESP – European Society of Pathology EurocanPlatform French BBMRI Node German BBMRI Node German Society of Pathology Italian BBMRI Node Medical University of Graz OECI – Organisation of European Cancer Institutes Royal College Pathologists
www.esp-pathology.org
http://eurocanplatform.eu/ www.biobanques.eu
forthcoming www.dgp-berlin.de
www.bbmri-eric.it
www.meduni-graz.at
www.oeci.eu
www.rcpath.org
WHITE PAPER http://www.impactsnetwork.eu/Sections.aspx?section=170
Solutions
:
-RSS new study designs -Networks for accessibility of AT -Standardization of analysis methods
G. Stanta
Solutions
:
-RSS new study designs -Networks for accessibility of AT -Standardization of analysis methods
G. Stanta
RETROSPECTIVE SURVIVAL STUDIES (RSS)
STUDY DESIGN: WELL DEFINED AT THE BEGINNING OF THE STUDY CASES: POPULATION BASED or HIGH NUMBER UNSELECTED CASES COLLECTION OF CASES: WITHOUT FOLLOW-UP DATA MOLECULAR ANALYSES:
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ACCURATE MICRODISSECTION -DEFINED SOPs AND IQCs OUTCOMES: PROSPECTIVELY LIKE COLLECTED POSITIVE RESULTS NEGATIVE RESULTS ADDITIONAL STUDIES IN DIFFERENT POPULATION PROCESS OF VERIFICATION AND VALIDATION
G. Stanta
Solutions
:
-RSS new study designs -Networks for accessibility of AT -Standardization of analysis methods
G. Stanta
MODELS OF CLINICAL DATA AND TISSUE COLLECTION
Cancer Inst Pathology Pathology Network
Hospital Clinical Information
OECI MODEL
Archive of FFPE Tissues Hospital Clinical Information Archive of FFPE Tissues
BBMRI MODEL
Tumor Registry
Hospital
Pathology
Hospital Clinical Information Archive of FFPE Tissues
EPAAC MODEL
Regional Organiz.
Hospital Pathology
Hospital Clinical Information
AREA MODEL
Archive of FFPE Tissues
G. Stanta
SIAPEC
NIPAB Network of Italian Pathology Archive Biobanks Pathology Departments joined to develop a national network Tissue paraffin blocks stored for a minimum of 20 years by law.
It is estimated that in pathology archives over 300 million cases and around one billion tissue specimens are stored
How to organize the network?
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The network is set up as a virtual network of pathology archives (samples + associated clinical data) for clinical research.
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Participation in the network and in the projects is voluntary and collaborative (these materials are residues from clinical procedures with specific requirements).
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Clinical and follow-up information can be directly collected by pathologists because they also deal with the diagnostic procedures.
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Privacy is guaranteed by the professional secrecy of pathologists, who have the duty to pseudo-anonymize the cases.
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There can be different network models for different strategies that can be embedded in BBMRI-ERIC.
Solutions
:
-RSS new study designs -Networks for accessibility of AT -Standardization of analysis methods
G. Stanta
Clinical research irreproducibility
SOURCES OF VARIABILITY # Tissue and macromolecule pre-analytical preservation # Selection and standardization of analytical procedures # Heterogeneity at the clinical, morphological or molecular level Technological complexity Biological complexity BIOLOGICAL COMPLEXITY
G. Stanta
Clinical research irreproducibility
SOURCES OF VARIABILITY # Tissue and macromolecule pre-analytical preservation # Selection and standardization of analytical procedures # Heterogeneity at the clinical, morphological or molecular level Technological complexity Biological complexity BIOLOGICAL COMPLEXITY
G. Stanta
Pre-analytical Conditions in IHC
Outside the pathology lab Inside the pathology lab
G. Stanta
Pre-analytical Conditions in IHC
Outside the pathology lab Inside the pathology lab
Proteins during the preanalytical phase may be categorized into three groups: (1) predictable stable; (2) predictable unstable; (3) unpredictable.
RNA is always degraded and needs specific technical capabilities for extraction and degradation standardization.
CEN (the European Committee for Standardization) is developing preanalytic technical specifications on proteins, DNA and RNA in tissues to ISO 15189 which might become instrumental in 2015.
G. Stanta
Clinical research irreproducibility
SOURCES OF VARIABILITY # Tissue and macromolecule pre-analytical preservation # Selection and standardization of analytical procedures # Heterogeneity at the clinical, morphological or molecular level Technological complexity Biological complexity BIOLOGICAL COMPLEXITY
G. Stanta
Bonin S , Stanta G. Nucleic acids extraction methods in fixed and paraffin embedded tissues in cancer diagnostics. Exp Rev Mol. Diagn. 2013,13.
SOURCES OF VARIABILITY # Selection and standardization of analytical procedures
Serena Bonin, Falk Hlubek, Jean Benhattar, Carsten Denkert, Manfred Dietel, Pedro L. Fernandez, Gerald Höfler, Hannelore Kothmaier, Bozo Kruslin, Chiara Maria Mazzanti, Aurel Perren, Helmuth Popper, Aldo Scarpa, Paula Soares, Giorgio Stanta and Patricia JTA Groenen.”MULTICENTRE VALIDATION STUDY OF NUCLEIC ACIDS EXTRACTION FROM FFPE TISSUES” Virchow Arch 2009 “
Reverse transcription yield, indeed, can vary up to 100-fold depending on priming strategy, on the used enzyme, on the starting quantity of target RNA and even on the type of sequence that is going to be detected.
”
G. Stanta
Bonin S , Stanta G. Nucleic acids extraction methods in fixed and paraffin embedded tissues in cancer diagnostics. Exp Rev Mol. Diagn. 2013,13.
SOURCES OF VARIABILITY # Selection and standardization of analytical procedures
Serena Bonin, Falk Hlubek, Jean Benhattar, Carsten Denkert, Manfred Dietel, Pedro L. Fernandez, Gerald Höfler, Hannelore Kothmaier, Bozo Kruslin, Chiara Maria Mazzanti, Aurel Perren, Helmuth Popper, Aldo Scarpa, Paula Soares, Giorgio Stanta and Patricia JTA Groenen.”MULTICENTRE VALIDATION STUDY OF NUCLEIC ACIDS EXTRACTION FROM FFPE TISSUES” Virchow Arch 2009
SOPs-IQCs!!!
strategy, on the used enzyme, on the starting quantity of target RNA and even on the type of sequence that is going to be detected.
”
G. Stanta
Section Lysate
Proteomics in archive tissues
KF Becker
Clinical research irreproducibility
SOURCES OF VARIABILITY # Tissue and macromolecule pre-analytical preservation # Selection and standardization of analytical procedures # Heterogeneity at the clinical, morphological or molecular level Technological complexity Biological complexity BIOLOGICAL COMPLEXITY
G. Stanta
The complex problem of heterogeneity
MACROSCOPIC HETEROGENEITY
ETHNIC HETEROGENEITY CLINICAL HETEROGENEITY
MICROSCOPIC TISSUE HETEROGENEITY
HISTOLOGIC TISSUE COMPOSITION TISSUE REACTION
MOLECULAR HETEROGENEITY
GENETIC CLONAL EVOLUTION EPIGENETIC CLONAL EVOLUTION PHENOTYPIC PLASTICITY HOMO/HETERO-TYPIC INTERACTIONS
The complex problem of heterogeneity
MACROSCOPIC HETEROGENEITY
ETHNIC HETEROGENEITY CLINICAL HETEROGENEITY Design of the study
MICROSCOPIC TISSUE HETEROGENEITY
HISTOLOGIC TISSUE COMPOSITION TISSUE REACTION
MOLECULAR HETEROGENEITY
GENETIC CLONAL EVOLUTION EPIGENETIC CLONAL EVOLUTION PHENOTYPIC PLASTICITY HOMO/HETERO-TYPIC INTERACTIONS Micro-dissection
The complex problem of heterogeneity
MACROSCOPIC HETEROGENEITY
ETHNIC HETEROGENEITY CLINICAL HETEROGENEITY Design of the study
MICROSCOPIC TISSUE HETEROGENEITY
HISTOLOGIC TISSUE COMPOSITION TISSUE REACTION
MOLECULAR HETEROGENEITY
GENETIC CLONAL EVOLUTION EPIGENETIC CLONAL EVOLUTION PHENOTYPIC PLASTICITY HOMO/HETERO-TYPIC INTERACTIONS Micro-dissection NGS In situ methods
MICROSCOPIC TISSUE HETEROGENEITY
T.Centre
T. Invasion Hlubek et al.,Int J Cancer, 2007 F Elloumi et al BMC Medical Genomics 4:54;2011
G. Stanta
TMA ARRAYER MICRODISSECTION GENE - EXPRESSION QUANTITATIVE ANALYSIS - Ct Gene β-Actin mRNA CDK2 mRNA Sample Coring 1 Coring 2 Coring 1 Coring 2 1 2
21.48
28.45
21.64
28.22
29.43
32.92
29.16
32.92
3 4
23.71
28.84
23.72
28.75
32.32
33.29
31.99
33.29
5
28.08
28.36
33.24
33.24
#Treatment after coring 50°C for 30 min plus 60°C for 10 min (especially for 5mm cores) #Expected RNA yield from 5 sections (1cm 2 ), 5 μm thick: 5 - 25 μg (related to tissue type and extraction method
)
# 1 TM A #1
Gene β-Actin mRNA Sample Tissues Coring 1
23.01* 21.64
2
28.48
28.22
CDK2 mRNA Tissues Coring
30.11
29.16
33.13
32.92
3 4
24.53
29.72
23.72
28.75
31.76
33.25
31.99
33.29
5
29.15
28.36
33.56
33.24
*Cts after real time amplification of 10 ng of cDNA after reverse transcription with random hexamers - not standardized Cts G. Stanta
“Garbage in garbage out” “Standard in standard out”
G. Stanta