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

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Presentation on theme: "High Resolution Data from Archive Tissue Analysis Malpensa 6 November 2012 Giorgio Stanta, Medical Sciences Department University of Trieste."— Presentation transcript:

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

2 SYSTEMS PATHOLOGY Application Confirmation Bench to Bedside Translational Research Reverse Translational Research Bedside to Bench CELL CULTURE ANIMAL MODELS FRESH TISSUES ARCHIVE TISSUES Deep sequencing TRANSLATIONAL AND REVERSE TRANSLATIONAL RESEARCH

3 ARCHIVE TISSUES: IMPROVING MOLECULAR MEDICINE RESEARCH AND CLINICAL PRACTICE

4 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.

5 DIAGNOSTICS SURGERY PATHOLOGY Selection Fixation Paraffin- embedding Slides preparation Diagnosis PATHOLOGY ARCHIVES HUMAN TISSUES DIAGNOSTIC FLOW Surgical Left-ove r

6 #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 PATHOLOGY Selection Fixation Paraffin- embedding Slides preparation Diagnosis PATHOLOGY ARCHIVES HUMAN TISSUES DIAGNOSTIC FLOW Surgical Left-ove r

7 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

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

9 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.

10 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.

11 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.

12 RESEARCH IN ARCHIVE TISSUES OPPORTUNITIES: #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) #.................................................

13 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)

14 Pathology 1 Hospital Clinical Information Archive of FFPE Tissues Pathology 2 Hospital Clinical Information Archive of FFPE Tissues Pathology 3 Hospital Clinical Information Archive of FFPE Tissues Pathology 4 Hospital Clinical Information Archive of FFPE Tissues Pathology 5 Hospital Clinical Information Archive of FFPE Tissues CLINICAL RESEARCH IN AT

15 Pathology 1 Hospital Clinical Information Archive of FFPE Tissues Pathology 2 Hospital Clinical Information Archive of FFPE Tissues Pathology 3 Hospital Clinical Information Archive of FFPE Tissues Pathology 4 Hospital Clinical Information Archive of FFPE Tissues Pathology 5 Hospital Clinical Information Archive of FFPE Tissues CLINICAL RESEARCH IN AT

16 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

17 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

18 Clinical and Tissue Heterogeneity A-CLINICAL HETEROGENEITY: related to different patient conditions (different tumor type, age, systemic diseases, etc.)

19 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)

20 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

21 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.

22 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.

23 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 MODEL FOR INCOMPLETE PENETRANCE OF MUTATIONS The Genomic Landscapes of Breast and Colon Cancers, Wood et al., Science 2007.

24 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) MODEL FOR INCOMPLETE PENETRANCE OF MUTATIONS The Genomic Landscapes of Breast and Colon Cancers, Wood et al., Science 2007.

25 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…) MODEL FOR INCOMPLETE PENETRANCE OF MUTATIONS The Genomic Landscapes of Breast and Colon Cancers, Wood et al., Science 2007.

26 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 MODEL FOR INCOMPLETE PENETRANCE OF MUTATIONS The Genomic Landscapes of Breast and Colon Cancers, Wood et al., Science 2007.

27 Heterogeneity is the major biological problem as source of a complex variability The technical solutions are:

28 Heterogeneity is the major biological problem as source of a complex variability The technical solutions are: 1-Tissue selection by micro-dissection

29 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

30 TMA#1 TISSUE-ARRAYER as MICRODISSECTOR

31 Core diametre (mm)Core surface (mm2)Sections for 1 cm 2 37.06514 519.625 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 (1cm 2 ), 5 μm thick: 5 - 25 μg (related to tissue type and extraction method)

32 Geneβ-ActinCDK2 Tissues Coring only Coring + treatment Tissues Coring only Coring + treatment Sample 123.01*21.4821.6430.1129.4329.16 228.4828.4528.2233.1332.92 324.5323.7123.7231.7632.3231.99 429.7228.8428.7533.2533.29 529.1528.0828.3633.5633.24 GENE EXPRESSION QUANTITATIVE ANALYSIS - Ct *Real Time amplification of 10 ng of cDNA after reverse transcription with random hexamers - not standardized Cts

33 PROTEIN EXTRACTION -5 sections of 10 μm from 5mm cores for a total surface of 1 cm 2, 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.

34 #1 TMA #1 IHC

35 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

36 PREANALYTICAL PRESERVATION OF ARCHIVE TISSUES PATHOLOGY DEPARTMENT D Fixation E Grossing F Embedding G Archive SURGERY A-B Warm Ischemia HOSPITAL ORG C Transport to A – B sec - hs C – D hs - days D – F hs - days G - years Vacuum transport Time control PROBLEMS

37 PREANALYTICAL PRESERVATION OF ARCHIVE TISSUES PATHOLOGY DEPARTMENT D Fixation E Grossing F Embedding G Archive SURGERY A-B Warm Ischemia HOSPITAL ORG C Transport to A – B sec - hs C – D hs - days D – F hs - days G - years Control of inducible genes Exhaustive dehydration Temperature control Early grossing Dark room Control of temperature and humidity Vacuum transport Time control New fixatives PROBLEMS SOLUTIONS FIXATION AT LOW TEMPERATURE

38 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

39 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 FIXATION TIME RATIO

40 DIAGNOSTICS SURGERY PATHOLOGY Selection Fixation Paraffin- embedding Slides preparation Diagnosis PATHOLOGY ARCHIVES HUMAN TISSUES DIAGNOSTIC FLOW TISSUE FIXATION Standardized time of fixation NewFix Fresh Tissue NEW FORMALIN-FREE FIXATIVES

41

42 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

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

44 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).. Method standardization mRNA Protein

45 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. Method standardization mRNA Protein

46 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. Method standardization mRNA Protein

47 POSSIBLE SOLUTIONS #Method standardization #Commercial kits #Robotics #Quality assessment #Laboratory certification #....................................

48 Proteomics in archival tissues Section Lysate K. Becker

49 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 ……………….. > …………………………………………… > ………………………………………..

50 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

51 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 OPERATORThe time can be very CLINICIANS AS USERSshort even less PATIENTS TO BE BENEFITEDthan 1 year


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