Le Biobanche di Campioni Inclusi Bari, 16/12/14 Giorgio Stanta, Dipartimento di Scienze Mediche -...

Le Biobanche di Campioni Inclusi

Bari, 16/12/14

Giorgio Stanta, Dipartimento di Scienze Mediche - Università di Trieste

MOLECULAR PATHOLOGY WGBIOBANKING AND MOLECULAR PATHOBIOLOGY WG

ARCHIVE TISSUES: IMPROVING MOLECULAR MEDICINE RESEARCH AND CLINICAL PRACTICEwww.impactsnetwork.eu

G. Stanta

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

MOLECULAR PATHOLOGY WG

BIOBANKING AND MOLECULAR PATHOBIOLOGY WG

BBMRI-ERIC CLINICAL BIOBANKS MEETINGBerlin 17/09/14

G. Stanta

ARCHIVE TISSUES WG

CLINICAL BIOBANKING WG

MEDICO-BIOLOGICAL RESEARCH

PATI

ENTS

BASIC RESEARCH………………

TRANSLATIONAL RESEARCH

RETROSPECTIVE CLINICAL RESEARCH

> 10 years on average > short timedevoted to future patients 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

G. Stanta

Applied medicine and clinical research are today an integrated and indissoluble process

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

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

Gene expression signatures for predicting the outcome in stage II colorectal cancer meta-analysis

Participating Organizations Organization SiteAustrian BBMRI Node www.bbmri.at

BBMRI-ERIC – Biobanking and Biomolecular Resources Research Infrastructure- European Research Infrastructure Consortium

www.bbmri-eric.eu

Czech BBMRI Node www.recamo.cz/en/bbmri

EATRIS – European Infrastructure for Translational Medicine

www.eatris.eu

ECCO – European CanCer Organisation

www.ecco-org.eu

ECPC – European Cancer Patient Coalition

www.ecpc.eu

EPAAC – European Partnership for Action Against Cancer

www.epaac.eu

ESP – European Society of Pathology

www.esp-pathology.org

EurocanPlatform http://eurocanplatform.eu/

French BBMRI Node www.biobanques.eu

German BBMRI Node forthcoming

German Society of Pathology www.dgp-berlin.de

Italian BBMRI Node www.bbmri-eric.it

Medical University of Graz www.meduni-graz.at

OECI – Organisation of European Cancer Institutes

www.oeci.eu

Royal College Pathologists 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

RETROSPECTIVE SURVIVAL STUDIES (RSS) CASES: POPULATION BASED or HIGH NUMBER UNSELECTED CASES

STUDY DESIGN: WELL DEFINED AT THE BEGINNING OF THE STUDY

COLLECTION OF CASES: WITHOUT FOLLOW-UP DATA

MOLECULAR ANALYSES: -ACCURATE MICRODISSECTION -DEFINED SOPs AND IQCs

OUTCOMES: PROSPECTIVELY-LIKE COLLECTED

NEGATIVE RESULTSPOSITIVE RESULTS

ADDITIONAL STUDIES IN DIFFERENT POPULATION

PROCESS OF VERIFICATION AND VALIDATION G. Stanta


G. Stanta

MODELS OF CLINICAL DATA AND TISSUE COLLECTION

G. Stanta

RETR

OSP

ECTI

VE S

URV

IVAL

STU

DIE

S

Hospital Clinical Information

Archive of FFPE Tissues

TumorRegistry

Hospital

Pathology

EPAACMODEL

PathologyNetwork



BBMRIMODEL



RegionalOrganiz.

Hospital

Pathology

AREAMODEL



Cancer Inst

Pathology

OECIMODEL

NIPABNetwork 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

SIAPEC

How to organize the network?#The network is set up as a virtual network of pathology archives (samples + associated clinical data) for clinical research.

# Participation in the network and in the projects is voluntary and collaborative (these materials are residues from clinical procedures with specific requirements).

# Clinical and follow-up information can be directly collected by pathologists because they also deal with the diagnostic procedures.

# Privacy is guaranteed by the professional secrecy of pathologists, who have the duty to pseudo-anonymize the cases.

# There can be different network models for different strategies that can be embedded in BBMRI-ERIC.


G. Stanta

G. Stanta

Clinical research irreproducibility

Biological complexity

Technological complexity

SOURCES OF VARIABILITY #Tissue and macromolecule pre-analytical preservation

#Selection and standardization of analytical procedures

#Heterogeneity at the clinical, morphological or molecular level

BIOLOGICAL COMPLEXITY

Pre-analytical Conditions in IHC

G. Stanta

Outside the pathology lab Inside the pathology lab

Pre-analytical Conditions in IHC

G. Stanta

Outside the pathology lab Inside the pathology labProteins 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








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

SOURCES OF VARIABILITY #Selection and standardization of analytical procedures

Bonin S , Stanta G. Nucleic acids extraction methods in fixed and paraffin-embedded tissues in cancer diagnostics. Exp Rev Mol. Diagn. 2013,13.

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

SOURCES OF VARIABILITY #Selection and standardization of analytical procedures

Bonin S , Stanta G. Nucleic acids extraction methods in fixed and paraffin-embedded tissues in cancer diagnostics. Exp Rev Mol. Diagn. 2013,13.

SOPs-IQCs!!!

Proteomics in archive tissues

Section

Lysate

KF Becker

G. Stanta








The complex problem of heterogeneityMACROSCOPIC HETEROGENEITYETHNIC HETEROGENEITYCLINICAL HETEROGENEITY

MICROSCOPIC TISSUE HETEROGENEITYHISTOLOGIC TISSUE COMPOSITIONTISSUE REACTION

MOLECULAR HETEROGENEITYGENETIC CLONAL EVOLUTION EPIGENETIC CLONAL EVOLUTIONPHENOTYPIC PLASTICITYHOMO/HETERO-TYPIC INTERACTIONS




Design of the study

Micro-dissection




Design of the study

Micro-dissection

NGS

In situ methods

T.Centre T. Invasion

Hlubek et al.,Int J Cancer, 2007 F Elloumi et al BMC Medical Genomics 4:54;2011

G. Stanta

MICROSCOPIC TISSUE HETEROGENEITY

TMA ARRAYER MICRODISSECTION GENE - EXPRESSION QUANTITATIVE ANALYSIS - CtGene β-Actin mRNA CDK2 mRNASample Coring 1 Coring 2 Coring 1 Coring 2

1 21.48 21.64 29.43 29.162 28.45 28.22 32.92 32.92

3 23.71 23.72 32.32 31.994 28.84 28.75 33.29 33.295 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 (1cm2), 5 μm thick: 5 - 25 μg (related to tissue type and extraction method)

#1

TMA

#1

Gene β-Actin mRNA CDK2 mRNASample Tissues Coring Tissues Coring

1 23.01* 21.64 30.11 29.16

2 28.48 28.22 33.13 32.92

3 24.53 23.72 31.76 31.99

4 29.72 28.75 33.25 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

Le Biobanche di Campioni Inclusi Bari, 16/12/14 Giorgio Stanta, Dipartimento di Scienze Mediche -...

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