High Resolution Data from Archive Tissue Analysis Malpensa 6 November 2012 Giorgio Stanta, Medical Sciences Department University of Trieste.

“High Resolution Data from Archive Tissue Analysis”

Malpensa6 November 2012

Giorgio Stanta, Medical Sciences Department University of Trieste

SYSTEMS PATHOLOG

Y

Application

Confirmation

Bench to BedsideTranslational Research

Reverse Translational ResearchBedside to Bench

CELL

CU

LTU

RE

ANIM

AL M

OD

ELS

FRESH TISSU

ES

ARCHIVE TISSU

ES

Deep sequencing

TRANSLATIONAL ANDREVERSE TRANSLATIONAL RESEARCH

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

SURGERY

PATHOLOGYSelection

FixationParaffin-embedding

Slidespreparation

Diagnosis

PATHOLOGYARCHIVES

HUMAN TISSUESDIAGNOSTIC FLOW

Surgical Left-over

Surgical Left-over

#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.

DIAGNOSTICS

SURGERY

PATHOLOGYSelection


Slidespreparation

Diagnosis

PATHOLOGYARCHIVES


Surgical Left-over

Surgical Left-over

BIOBANKING AND MOLECULAR MOLECULAR PATHOLOGY AND PATHOBIOLOGY W.G. 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

NETWORKING

AVAILABILITY OF ARCHIVE TISSUES #Where to find those tissuesThe OECI – ESP organizations represent almost all available archive tissues in Europe.

AVAILABILITY OF ARCHIVE TISSUES #Where to find those tissuesThe OECI – ESP organizations represent almost all available archive tissues in Europe. #How to involve pathologistsVoluntary and collaborative participants in the specific project are required.

AVAILABILITY OF ARCHIVE TISSUES #Where to find those tissuesThe OECI – ESP organizations represent almost all available archive tissues in Europe. #How to involve pathologistsVoluntary and collaborative participants in the specific project are required.#How to obtain follow-up dataWe 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 tissuesThe OECI – ESP organizations represent almost all available archive tissues in Europe. #How to involve pathologistsVoluntary and collaborative participants in the specific project are required.#How to obtain follow-up dataWe 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 BBPathology 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 TISSUESOPPORTUNITIES:#Preliminary retrospective research (lower costs, some level of warranty for subsequent prospective studies)

#Availability of complete clinical heterogeneity and even of rare entities#Same tissues as those available for molecular diagnosis in patients#High level of clinical information#Possibility of further information (also after the conclusion of the study)

#Histological review and further histological data (new molecular classification …..)

#................................................

PROBLEMS:#Degradation of macromolecules especially RNA#Possible selection bias common in retrospective studies#New/old ethical problems related to sensitive data and type of consent#Necessity to maintain significant quantities of tissues for future diagnostic procedure#Non-standardized methods of analysis (very few laboratories have 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)

Pathology 1Hospital Clinical Information

Archive of FFPE Tissues









CLINICALRESEARCH

IN AT











CLINICALRESEARCH

IN AT

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.)


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)


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


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 metastasesD-FUNCTIONAL HETEROGENEITY:-Genetic heterogeneity The Genomic Landscapes of Breast and

Colon Cancers‖, Wood et al., Science 2007.


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 metastasesD-FUNCTIONAL HETEROGENEITY:-Genetic heterogeneity-Epigenetic heterogeneity

The Genomic Landscapes of Breast and Colon Cancers‖, Wood et al., Science 2007.


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 metastasesD-FUNCTIONAL HETEROGENEITY:-Genetic heterogeneity-Epigenetic heterogeneity-Phenotypic heterogeneity

MODEL FOR INCOMPLETE PENETRANCE OF MUTATIONS



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 metastasesD-FUNCTIONAL HETEROGENEITY:-Genetic heterogeneity-Epigenetic heterogeneity-Phenotypic heterogeneity -Functionally defined heterogeneity (border or central tumor)




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 metastasesD-FUNCTIONAL HETEROGENEITY:-Genetic heterogeneity-Epigenetic heterogeneity-Phenotypic heterogeneity -Functionally defined heterogeneity (border or central tumor)-Stochastic heterogeneity (stochastic single-cell/molecule event…)




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 metastasesD-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 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 diametre (mm) Core surface (mm2) Sections for 1 cm2

3 7.065 14

5 19.62 5

CORE SIZE

#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 β-Actin CDK2

Tissues Coringonly

Coring +treatment Tissues Coring

onlyCoring +

treatmentSample

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

GENE EXPRESSION QUANTITATIVE ANALYSIS - Ct

*Real Time amplification of 10 ng of cDNA after reverse transcription with random hexamers - not standardized Cts

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

1 2 3 4 5 6 7

Tessuti originari

coring +trattamento

GAPDH

0

500000

1000000

1500000

2000000

2500000

3000000

1 2 3 4 5 6 7

Tessuti originari

coring +trattamento

CDK2

PROTEIN EXTRACTION

-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 .

#1TMA

#1

IHC





PREANALYTICAL PRESERVATION OF ARCHIVE TISSUES

PATHOLOGY DEPARTMENT

DFixation

EGrossing

FEmbedding

G Archive

SURGERY A-B Warm Ischemia

HOSPITAL ORGC

Transport to

A – B sec - hsC – D hs - days

D – F hs - daysG - years

Vacuum transportTime control

PRO

BLEM

S

PREANALYTICAL PRESERVATION OF ARCHIVE TISSUES

PATHOLOGY DEPARTMENT

DFixation

EGrossing

FEmbedding

G Archive

SURGERY A-B Warm Ischemia

HOSPITAL ORGC

Transport to

A – B sec - hsC – D hs - days

D – F hs - daysG - years

Control of inducible genes

Exhaustive dehydrationTemperature

control

Early grossing

Dark roomControl of temperature and humidity

Vacuum transportTime control

New fixatives

PRO

BLEM

SSO

LUTI

ON

S

FIXATION AT LOW TEMPERATURE

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

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

HYPOXIAFIXATION

TIME RATIO

DIAGNOSTICS

SURGERY

PATHOLOGY

Selection


Slidespreparation

Diagnosis

PATHOLOGYARCHIVES


TISSUEFIXATION

Standardized time of fixation

pH 4 7

NewFix

pH 4 7

Fresh Tissue

NEW FORMALIN-FREE FIXATIVES





PROBLEMS# Standardization should start from the type of the analyzed molecules, mRNA and proteins can give very different results.

.

Method standardizationmRNA Protein


# 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.




# 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.


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

……………….. > …………………………………………… > ………………………………………..

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).

OECI Philosophy: Care providers have a moral and legal obligation to protect the interests of their patients

PATIENT AS DONOR

RESEARCHER AS OPERATOR The time can be very

CLINICIANS AS USERS short even less

PATIENTS TO BE BENEFITED than 1 year

High Resolution Data from Archive Tissue Analysis Malpensa 6 November 2012 Giorgio Stanta, Medical Sciences Department University of Trieste.

Documents

available archive tissues

archive tissues opportunities

type of tissues

clinical practice slide

molecular molecular

clinical records

bb pathology archives

oeci esp organizations