“High Resolution Data from Archive
Tissue Analysis”
Malpensa
6 November 2012
Giorgio Stanta,
Medical Sciences Department University of Trieste
TRANSLATIONAL AND
REVERSE TRANSLATIONAL RESEARCH
Bench to Bedside
Translational Research
Deep sequencing
ANIMAL MODELS
Confirmation
Reverse Translational Research
Bedside to Bench
ARCHIVE TISSUES
SYSTEMS
PATHOLOGY
FRESH TISSUES
CELL CULTURE
Application
ARCHIVE TISSUES: IMPROVING MOLECULAR MEDICINE
RESEARCH AND CLINICAL PRACTICE
ARCHIVE TISSUES
-These are the only tissues available in any hospital
for patients.
- The pathology archives storing those tissues
represent the widest collection of clinical tissues
available with the entire clinical heterogeneity range.
-Today it is possible to perform any type of molecular
analysis on this type of tissues.
DIAGNOSTICS
PATHOLOGY
Selection
SURGERY
Diagnosis
Slides
preparation
Fixation
Paraffinembedding
Surgical
SurgicalLeft-over
Left-over
HUMAN TISSUES
DIAGNOSTIC FLOW
PATHOLOGY
ARCHIVES
DIAGNOSTICS
PATHOLOGY
Selection
SURGERY
Diagnosis
Slides
preparation
#Millions of residual human tissue specimens with any kind
of even rare diseases are stored often for decades in the
archives of hospitals.
#These tissues are related to clinical records and very often
to very developed health informatic systems with follow-up
and outcome information.
#The archives are run by pathologists that have access to all
the information and are bound by professional secrecy.
Fixation
Paraffinembedding
Surgical
SurgicalLeft-over
Left-over
HUMAN TISSUES
DIAGNOSTIC FLOW
PATHOLOGY
ARCHIVES
NETWORKING
BIOBANKING AND MOLECULAR
PATHOBIOLOGY W.G.
MOLECULAR PATHOLOGY AND
BIOBANKING W.G.
#Pre-analytical conditions (IMPACTS)
#Method standardization (IMPACTS)
#Quality assessment and laboratory certification (OECI - ESP
and collaboration with any interested EU organization)
#Laboratory Developed Techniques (OECI)
#Archive tissue biobanking network (OECI, ESP, BBMRI)
#Multicentric studies activation (OECI, ESP)
#Training (OECI, ESP)
#Networking
AVAILABILITY OF ARCHIVE TISSUES
#Where to find those tissues
The OECI – ESP organizations represent almost all
available archive tissues in Europe.
AVAILABILITY OF ARCHIVE TISSUES
#Where to find those tissues
The OECI – ESP organizations represent almost all
available archive tissues in Europe.
#How to involve pathologists
Voluntary and collaborative participants in the specific
project are required.
AVAILABILITY OF ARCHIVE TISSUES
#Where to find those tissues
The OECI – ESP organizations represent almost all
available archive tissues in Europe.
#How to involve pathologists
Voluntary and collaborative participants in the specific
project are required.
#How to obtain follow-up data
We have already started with mapping of specific EU areas
in which collection of follow-up data is possible.
AVAILABILITY OF ARCHIVE TISSUES
#Where to find those tissues
The OECI – ESP organizations represent almost all
available archive tissues in Europe.
#How to involve pathologists
Voluntary and collaborative participants in the specific
project are required.
#How to obtain follow-up data
We have already started with mapping of specific EU areas
in which collection of follow-up data is possible.
#When does a pathology archive take the function of a BB
Pathology archives take the function of a biobank when
personal data are treated with a double coding, activated
only for those cases included in a specific project.
RESEARCH IN ARCHIVE TISSUES
OPPORTUNITIES:
PROBLEMS:
#Preliminary retrospective research #Degradation of macromolecules
(lower costs, some level of warranty for
especially RNA
subsequent prospective studies)
#Possible selection bias common
#Availability of complete clinical
in retrospective studies
heterogeneity and even of rare
#New/old ethical problems related
entities
to sensitive data and type of
#Same tissues as those available for
consent
molecular diagnosis in patients
#Necessity to maintain significant
#High level of clinical information
quantities of tissues for future
#Possibility of further information
diagnostic procedure
(also after the conclusion of the study)
#Non-standardized methods of
#Histological review and further
analysis (very few laboratories have
histological data (new molecular
classification …..)
#................................................
experience in RNA and protein analysis)
#.................................................
DISCOVERY AND VALIDATION OF CLINICAL
BIOMARKERS AND THERAPY TARGETS IN AT
DISCOVERY (BM and target identification in retrospective
studies)
PRECLINICAL VALIDATION (in clinical tissue residues)
CLINICAL VALIDATION (retrospective studies, prospective
trials and technical setting)
CLINICAL USE (performance evaluation in clinical tissues)
Hospital Clinical Information
Pathology 1
Archive of FFPE Tissues
Hospital Clinical Information
Pathology 2
Archive of FFPE Tissues
CLINICAL
RESEARCH
IN AT
Hospital Clinical Information
Pathology 3
Archive of FFPE Tissues
Hospital Clinical Information
Pathology 4
Archive of FFPE Tissues
Hospital Clinical Information
Pathology 5
Archive of FFPE Tissues
Hospital Clinical Information
Pathology 1
Archive of FFPE Tissues
Hospital Clinical Information
Pathology 2
Archive of FFPE Tissues
CLINICAL
RESEARCH
IN AT
Hospital Clinical Information
Pathology 3
Archive of FFPE Tissues
Hospital Clinical Information
Pathology 4
Archive of FFPE Tissues
Hospital Clinical Information
Pathology 5
Archive of FFPE Tissues
SOURCES OF CLINICAL RESEARCH AND
DIAGNOSTICS VARIABILITY
#Heterogeneity at the clinical, morphological or
molecular level
#Tissue and macromolecule pre-analytical
preservation
#Selection and standardization of analytical
procedures
SOURCES OF CLINICAL RESEARCH AND
DIAGNOSTICS VARIABILITY
#Heterogeneity at the clinical, morphological or
molecular level
#Tissue and macromolecule pre-analytical
preservation
#Selection and standardization of analytical
procedures
Clinical and Tissue Heterogeneity
A-CLINICAL HETEROGENEITY: related to different patient
conditions (different tumor type, age, systemic diseases,
etc.)
Clinical and Tissue Heterogeneity
A-CLINICAL HETEROGENEITY: related to different patient
conditions (different tumor type, age, systemic diseases,
etc.)
B-TISSUE RELATED HETEROGENEITY:
-Related to tissue complexity (fibrosis, flogosis, necrosis,
normal residual tissues…)
-Related to histological heterogeneity (Different
histological pattern of the same tumor)
Clinical and Tissue Heterogeneity
A-CLINICAL HETEROGENEITY: related to different patient
conditions (different tumor type, age, systemic diseases,
etc.)
B-TISSUE RELATED HETEROGENEITY:
-Related to tissue complexity (fibrosis, flogosis, necrosis,
normal residual tissues…)
-Related to histological heterogeneity (Different
histological pattern of the same tumor)
C-MOLECULAR HETEROGENEITY BY CLONAL EVOLUTION:
-In the primary tumor
-Differences between primary tumor and metastasis
-Among different metastases
Clinical and Tissue Heterogeneity
A-CLINICAL HETEROGENEITY: related to different patient
conditions (different tumor type, age, systemic diseases,
etc.)
B-TISSUE RELATED HETEROGENEITY:
-Related to tissue complexity (fibrosis, flogosis, necrosis,
normal residual tissues…)
-Related to histological heterogeneity (Different
histological pattern of the same tumor)
C-MOLECULAR HETEROGENEITY BY CLONAL EVOLUTION:
-In the primary tumor
-Differences between primary tumor and metastasis
-Among different metastases
D-FUNCTIONAL HETEROGENEITY:
-Genetic heterogeneity
The Genomic Landscapes of Breast and
Colon Cancers‖, Wood et al., Science
2007.
Clinical and Tissue Heterogeneity
A-CLINICAL HETEROGENEITY: related to different patient
conditions (different tumor type, age, systemic diseases,
etc.)
B-TISSUE RELATED HETEROGENEITY:
-Related to tissue complexity (fibrosis, flogosis, necrosis,
normal residual tissues…)
-Related to histological heterogeneity (Different
histological pattern of the same tumor)
C-MOLECULAR HETEROGENEITY BY CLONAL EVOLUTION:
-In the primary tumor
-Differences between primary tumor and metastasis
-Among different metastases
D-FUNCTIONAL HETEROGENEITY:
-Genetic heterogeneity
-Epigenetic heterogeneity
The Genomic Landscapes of Breast and
Colon Cancers‖, Wood et al., Science
2007.
Clinical and Tissue Heterogeneity
A-CLINICAL HETEROGENEITY: related to different patient
conditions (different tumor type, age, systemic diseases,
etc.)
B-TISSUE RELATED HETEROGENEITY:
-Related to tissue complexity (fibrosis, flogosis, necrosis,
normal residual tissues…)
-Related to histological heterogeneity (Different
histological pattern of the same tumor)
C-MOLECULAR HETEROGENEITY BY CLONAL EVOLUTION:
-In the primary tumor
-Differences between primary tumor and metastasis
-Among different metastases
D-FUNCTIONAL HETEROGENEITY:
-Genetic heterogeneity
-Epigenetic heterogeneity
-Phenotypic heterogeneity
The Genomic Landscapes of Breast and
Colon Cancers‖, Wood et al., Science
2007.
MODEL FOR INCOMPLETE
PENETRANCE OF
MUTATIONS
Clinical and Tissue Heterogeneity
A-CLINICAL HETEROGENEITY: related to different patient
conditions (different tumor type, age, systemic diseases,
etc.)
B-TISSUE RELATED HETEROGENEITY:
-Related to tissue complexity (fibrosis, flogosis, necrosis,
normal residual tissues…)
-Related to histological heterogeneity (Different
histological pattern of the same tumor)
C-MOLECULAR HETEROGENEITY BY CLONAL EVOLUTION:
-In the primary tumor
-Differences between primary tumor and metastasis
-Among different metastases
D-FUNCTIONAL HETEROGENEITY:
-Genetic heterogeneity
-Epigenetic heterogeneity
-Phenotypic heterogeneity
-Functionally defined heterogeneity (border or central
tumor)
The Genomic Landscapes of Breast and
Colon Cancers‖, Wood et al., Science
2007.
MODEL FOR INCOMPLETE
PENETRANCE OF
MUTATIONS
Clinical and Tissue Heterogeneity
A-CLINICAL HETEROGENEITY: related to different patient
conditions (different tumor type, age, systemic diseases,
etc.)
B-TISSUE RELATED HETEROGENEITY:
-Related to tissue complexity (fibrosis, flogosis, necrosis,
normal residual tissues…)
-Related to histological heterogeneity (Different
histological pattern of the same tumor)
C-MOLECULAR HETEROGENEITY BY CLONAL EVOLUTION:
-In the primary tumor
-Differences between primary tumor and metastasis
-Among different metastases
D-FUNCTIONAL HETEROGENEITY:
-Genetic heterogeneity
-Epigenetic heterogeneity
-Phenotypic heterogeneity
-Functionally defined heterogeneity (border or central
tumor)
-Stochastic heterogeneity (stochastic single-cell/molecule
event…)
The Genomic Landscapes of Breast and
Colon Cancers‖, Wood et al., Science
2007.
MODEL FOR INCOMPLETE
PENETRANCE OF
MUTATIONS
Clinical and Tissue Heterogeneity
A-CLINICAL HETEROGENEITY: related to different patient
conditions (different tumor type, age, systemic diseases,
etc.)
B-TISSUE RELATED HETEROGENEITY:
-Related to tissue complexity (fibrosis, flogosis, necrosis,
normal residual tissues…)
-Related to histological heterogeneity (Different
histological pattern of the same tumor)
C-MOLECULAR HETEROGENEITY BY CLONAL EVOLUTION:
-In the primary tumor
-Differences between primary tumor and metastasis
-Among different metastases
D-FUNCTIONAL HETEROGENEITY:
-Genetic heterogeneity
-Epigenetic heterogeneity
-Phenotypic heterogeneity
-Functionally defined heterogeneity (border or central
tumor)
-Stochastic heterogeneity (stochastic single-cell/molecule
event…)
-Micro-environment heterogeneity
The Genomic Landscapes of Breast and
Colon Cancers‖, Wood et al., Science
2007.
MODEL FOR INCOMPLETE
PENETRANCE OF
MUTATIONS
“Heterogeneity is the major biological problem
as source of a complex variability”
The technical solutions are:
“Heterogeneity is the major biological problem
as source of a complex variability”
The technical solutions are:
1-Tissue selection by micro-dissection
“Heterogeneity is the major biological problem
as source of a complex variability”
The technical solutions are:
1-Tissue selection by micro-dissection
2-Choice of extractive or in situ methods related to the diagnostic
or research question to resolve
TMA#1
TISSUE-ARRAYER as
MICRODISSECTOR
CORE SIZE
Core diametre (mm) Core surface (mm2)
Sections for 1 cm2
3
7.065
14
5
19.62
5
#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)
GENE EXPRESSION QUANTITATIVE ANALYSIS - Ct
Gene
β-Actin
CDK2
Tissues
Coring
only
Coring +
treatment
Tissues
Coring
only
Coring +
treatment
1
23.01*
21.48
21.64
30.11
29.43
29.16
2
28.48
28.45
28.22
33.13
32.92
32.92
3
24.53
23.71
23.72
31.76
32.32
31.99
4
29.72
28.84
28.75
33.25
33.29
33.29
5
29.15
28.08
28.36
33.56
33.24
33.24
Sample
*Real Time amplification of 10 ng of cDNA after reverse transcription with random hexamers - not
standardized Cts
PROTEIN EXTRACTION
GAPDH
7000000
6000000
5000000
Tessuti originari
4000000
coring +
trattamento
3000000
2000000
1000000
0
1
2
3
4
5
6
7
CDK2
3000000
2500000
2000000
Tessuti originari
1500000
coring +
trattamento
1000000
500000
0
1
2
3
4
5
6
7
-5 sections of 10 μm from 5mm cores for a total surface of 1 cm2, compared with a similar surface of the original tissue. Extraction by Qproteome FFPE Tissue Kit.
-Total protein concentration by NanoPhotometer™.
- DotBlot: 10 μl of1:200 of protein solution spotted on membrane. Antibodies against GAPDH and CDK2. Developed by ECL on
Immobilon membrane. Analysis of the dots by Versadoc with ImageJ software .
IHC
#1
TMA
#1
SOURCES OF CLINICAL RESEARCH AND
DIAGNOSTICS VARIABILITY
#Heterogeneity at the clinical, morphological or
molecular level
#Tissue and macromolecule pre-analytical
preservation
#Selection and standardization of analytical
procedures
PREANALYTICAL PRESERVATION OF ARCHIVE TISSUES
PROBLEMS
SURGERY A-B
Warm Ischemia
HOSPITAL ORG
C
Transport to
PATHOLOGY DEPARTMENT
D
Fixation
E
Grossing
F
Embedding
A – B sec - hs
C – D hs - days
D – F hs - days
Vacuum transport
Time control
G
Archive
G - years
PREANALYTICAL PRESERVATION OF ARCHIVE TISSUES
PROBLEMS
SURGERY A-B
Warm Ischemia
HOSPITAL ORG
C
Transport to
PATHOLOGY DEPARTMENT
D
Fixation
E
Grossing
F
Embedding
A – B sec - hs
C – D hs - days
SOLUTIONS
D – F hs - days
G
Archive
G - years
Early grossing
Vacuum transport
Control of
inducible genes
Time
control
FIXATION AT
LOW
TEMPERATURE
New fixatives
Exhaustive
dehydration
Temperature
control
Dark room
Control of
temperature
and humidity
RNA DEGRADATION IN FORMALIN-FIXED CELLS BY FIXATION
TIME
I.Dotti, S.Bonin, G. Basili, E. Nardon, A. Balani, S. Siracusano, F. Zanconati, S. Palmisano, N. De Manzini and G. Stanta. “Effects of formalin, methacarn and
FineFIX fixatives on RNA preservation”. Diagn Mol Pathol 19:112-122; 2010
S Bonin, F Petrera, G Stanta, “PCR and RT-PCR Analysis in Archivial Postmortem Tissues” in “Encyclopedia of Medical Genomics and Proteomics” Marcel
Dekker, New York: 985-988; 2005
RNA DEGRADATION IN FORMALIN-FIXED CELLS BY FIXATION
TIME
HYPOXIA
TIME RATIO
FIXATION
I.Dotti, S.Bonin, G. Basili, E. Nardon, A. Balani, S. Siracusano, F. Zanconati, S. Palmisano, N. De Manzini and G. Stanta. “Effects of formalin, methacarn and
FineFIX fixatives on RNA preservation”. Diagn Mol Pathol 19:112-122; 2010
S Bonin, F Petrera, G Stanta, “PCR and RT-PCR Analysis in Archivial Postmortem Tissues” in “Encyclopedia of Medical Genomics and Proteomics” Marcel
Dekker, New York: 985-988; 2005
PATHOLOGY
DIAGNOSTICS
SURGERY
HUMAN TISSUES
DIAGNOSTIC FLOW
Selection
Fixation
Paraffinembedding
Slides
TISSUE
preparation FIXATION
Diagnosis
NEW FORMALIN-FREE FIXATIVES
pH 4
7
NewFix
pH 4
7
Fresh Tissue
Standardized
time of fixation
PATHOLOGY
ARCHIVES
SOURCES OF CLINICAL RESEARCH AND
DIAGNOSTICS VARIABILITY
#Heterogeneity at the clinical, morphological or
molecular level
#Tissue and macromolecule pre-analytical
preservation
#Selection and standardization of analytical
procedures
Method standardization
PROBLEMS
# Standardization should start from the type
of the analyzed molecules, mRNA and
proteins can give very different results.
.
mRNA
Protein
Method standardization
PROBLEMS
# Standardization should start from the type
of the analyzed molecules, mRNA and
proteins can give very different results.
# Using the same type of molecular
analysis, different methods can also give
different results, according to their
sensitivity or different quantitative
approach (IHC versus protein extractive
methods).
.
mRNA
Protein
Method standardization
PROBLEMS
# Standardization should start from the type
of the analyzed molecules, mRNA and
proteins can give very different results.
# Using the same type of molecular
analysis, different methods can also give
different results, according to their
sensitivity or different quantitative
approach (IHC versus protein extractive
methods).
# Several similar methods for the same type
of analysis can have very different
sensitivity and specificity and the
procedures need to be standardized.
mRNA
Protein
Method standardization
PROBLEMS
# Standardization should start from the type
of the analyzed molecules, mRNA and
proteins can give very different results.
# Using the same type of molecular
analysis, different methods can also give
different results, according to their
sensitivity or different quantitative
approach (IHC versus protein extractive
methods).
# Several similar methods for the same type
of analysis can have very different
sensitivity and specificity and the
procedures need to be standardized.
# One of the possibilities to standardize
methods is the use of commercial kits, but
these, especially for diagnostics, must be
widely validated at the lab level.
mRNA
Protein
POSSIBLE SOLUTIONS
#Method standardization
#Commercial kits
#Robotics
#Quality assessment
#Laboratory certification
#....................................
Proteomics in archival tissues
Section
Lysate
K. Becker
EVALUATION OF TYPES OF ANALYSIS IN
ARCHIVE TISSUES
DNA > Qualitative analysis > Sequencing > High level of reproducibility
Microsatellite instability >Standardized procedures > High level of reproducibility
Promoter methylation >High number of non-standardized methods (MGMT, CIMP)
RNA >Quantitative analysis > RT- and qRealTime PCR >few experienced labs
MicroRNAs >standardization in development >good results in AT
Long non-coding RNAs >evaluation in development
IHC >Mature technology >Established specificity and quality for diagnostic Ab
>Experienced lab for specificity and setting of new Ab
Proteomics > very promising technologies, but still not diffused experience
……………….. > …………………………………………… > ………………………………………..
OECI Philosophy: Care providers have a moral and legal obligation to protect the
interests of their patients
Laboratory Developed Techniques (LDT)
Development of definition and rules on Laboratory Developed Techniques (LDT) and the new
concept of Clinical Research Use Only (CRUO)
There is the necessity to accelerate clinical application in well-prepared and developed
institutions of new effective increasingly available biomarkers, but still not approved by
regulatory institutions. This will help a high number of patients to benefit from it before its
approved commercial use. This will also accelerate clinical performance evaluation. Of course
specific bioethical, training and organizational rules must be developed (institutions with
biotechnologists, pathologists and oncologists qualified for this activity).
OECI Philosophy: Care providers have a moral and legal obligation to protect the
interests of their patients
Laboratory Developed Techniques (LDT)
Development of definition and rules on Laboratory Developed Techniques (LDT) and the new
concept of Clinical Research Use Only (CRUO)
There is the necessity to accelerate clinical application in well-prepared and developed
institutions of new effective increasingly available biomarkers, but still not approved by
regulatory institutions. This will help a high number of patients to benefit from it before its
approved commercial use. This will also accelerate clinical performance evaluation. Of course
specific bioethical, training and organizational rules must be developed (institutions with
biotechnologists, pathologists and oncologists qualified for this activity).
PATIENT AS DONOR
RESEARCHER AS OPERATOR
CLINICIANS AS USERS
PATIENTS TO BE BENEFITED
The time can be very
short even less
than 1 year