dr dinah parums. the role of the pathologist in target identification and validation in targeted...
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THE ROLE OF THE PATHOLOGIST IN DRUG DISCOVERY AND DEVELOPMENT
- TARGET VALIDATIONDr Dinah Parums, Principal Pathologist
THE ROLE OF THE PATHOLOGIST IN DRUG DISCOVERY AND DEVELOPMENT
- TARGET VALIDATIONDr Dinah Parums, Principal Pathologist
Biotechnology and pharmaceutical companies are challenged to validate the pool of potential drug targets and determine those most appropriate to enter a drug development programme.
A valuable method of target validation is their localisation to specific cells and tissues using immunohistochemistry (IHC) and non isotopic in situ hybridisation (NISH) techniques pinpointing the expression of protein and nucleic acids respectively.
Biotechnology and pharmaceutical companies are challenged to validate the pool of potential drug targets and determine those most appropriate to enter a drug development programme.
A valuable method of target validation is their localisation to specific cells and tissues using immunohistochemistry (IHC) and non isotopic in situ hybridisation (NISH) techniques pinpointing the expression of protein and nucleic acids respectively.
IMMUNOHISTOCHEMISTRY and IMMUNOFLUORESCENCE
IMMUNOHISTOCHEMISTRY and IMMUNOFLUORESCENCE
Tissue sections from normal and diseased specimens on glass slides as whole sections, multiblocks or TMAs
Tissues are frozen or formalin fixed and embedded in paraffin wax
Formalin fixed tissues offer better morphology and are more readily available but fixation must be standardised
Tissue sections from normal and diseased specimens on glass slides as whole sections, multiblocks or TMAs
Tissues are frozen or formalin fixed and embedded in paraffin wax
Formalin fixed tissues offer better morphology and are more readily available but fixation must be standardised
WHAT CAN Immunohistochemistry (IHC) SHOW ? ?
WHAT CAN Immunohistochemistry (IHC) SHOW ? ?
The detection of target antigens (usually proteins) within tissues and cells
Relative level of target expression
Subcellular localisation of the target (nuclear, cytoplasmic, cell membrane)
The detection of target antigens (usually proteins) within tissues and cells
Relative level of target expression
Subcellular localisation of the target (nuclear, cytoplasmic, cell membrane)
McAb ASMA in myofibroblasts in healing skin
McAb ASMA in myofibroblasts in healing skinConfocal immunofluorescence
WHAT CAN IHC (and IMMUNOFLUORESCENCE) SHOW ?
WHAT CAN IHC (and IMMUNOFLUORESCENCE) SHOW ?
Double confocal
immunofluorescence
McAb CD31 localizing
to endothelial cells (Red)
McAb ASMA localizing
to smooth muscle cells (Green)
CONSIDERATIONS FOR ANTIBODY USECONSIDERATIONS FOR ANTIBODY USE
‘Clean’ monoclonal and polyclonal antibodies should be used (confirmed by western blot or immunoprecipitation)
Polyclonal antibodies should be affinity purified Antibodies generated from peptides or complete
proteins can be used Binding of an antibody to a target in tissues is empirical
thus each antibody should be tested separately for reactivity in tissues
‘Clean’ monoclonal and polyclonal antibodies should be used (confirmed by western blot or immunoprecipitation)
Polyclonal antibodies should be affinity purified Antibodies generated from peptides or complete
proteins can be used Binding of an antibody to a target in tissues is empirical
thus each antibody should be tested separately for reactivity in tissues
Polyclonal antibody to TGF beta in infiltrating lobular carcinoma of the breast localises to stromal spindle cells and collagen.
Immunoperoxidase with DAB.
Is this specific or not ?
6 KEY STEPS FOR IHC IN TUMOUR BIOMARKER DEVELOPMENT
1 NEED FOR SPECIFIC ANTIBODY
TO IDENTIFY PROTEIN MARKER.
2 TEST FOR SPECIFICITY IN HUMAN/ANIMAL TISSUE.
3 TEST FOR EFFECT OF CANDIDATE DRUG (CD) ON PROTEIN EXPRESSION IN TUMOUR XENOGRAFT IN ANIMAL MODEL.
4 FEASIBILITY AND VARIABILITY ASSESSMENT IN NORMAL AND HUMAN TUMOUR TISSUE (fresh, frozen or formalin-fixed).
5 EFFECT OF CD ON NORMAL AND TUMOUR TISSUE (fresh, frozen or formalin-fixed). PROOF OF MECHANISM/PROOF OF PRINCIPLE.
6 EFFECT OF CD ON NORMAL AND TUMOUR TISSUE (fresh frozen or formalin fixed) AND LINKED TO DISEASE OUTCOME. PROOF OF CONCEPT.
1 NEED FOR SPECIFIC ANTIBODY
TO IDENTIFY PROTEIN MARKER.
2 TEST FOR SPECIFICITY IN HUMAN/ANIMAL TISSUE.
3 TEST FOR EFFECT OF CANDIDATE DRUG (CD) ON PROTEIN EXPRESSION IN TUMOUR XENOGRAFT IN ANIMAL MODEL.
4 FEASIBILITY AND VARIABILITY ASSESSMENT IN NORMAL AND HUMAN TUMOUR TISSUE (fresh, frozen or formalin-fixed).
5 EFFECT OF CD ON NORMAL AND TUMOUR TISSUE (fresh, frozen or formalin-fixed). PROOF OF MECHANISM/PROOF OF PRINCIPLE.
6 EFFECT OF CD ON NORMAL AND TUMOUR TISSUE (fresh frozen or formalin fixed) AND LINKED TO DISEASE OUTCOME. PROOF OF CONCEPT.
Custom made
Commercially available
Academic institution
NON ISOTOPIC IN SITU HYBRIDIZATION (NISH) STUDIES
NON ISOTOPIC IN SITU HYBRIDIZATION (NISH) STUDIES
ISH assays allow for the detection of nucleic acid sequences in cells and tissues.
The target molecules are specific mRNA sequences RNA and DNA probes can be used For riboprobes it is common to make both sense and antisense
probes The antisense probe is a complementary strand to the target
mRNA and should bind with it Generally, riboprobes are chosen rather than oligonucleotide or
DNA probes The ideal probe length is debatable but 200 to 500 bases work
well. Longer probes may be more sensitive but can be difficult to
hybridize to their targets Radioisotopically labelled probes are very sensitive but yield
lower resolution with results being more difficult to assess Biotinylated probes may be problematic due to endogenous biotin
and biotin-binding proteins
ISH assays allow for the detection of nucleic acid sequences in cells and tissues.
The target molecules are specific mRNA sequences RNA and DNA probes can be used For riboprobes it is common to make both sense and antisense
probes The antisense probe is a complementary strand to the target
mRNA and should bind with it Generally, riboprobes are chosen rather than oligonucleotide or
DNA probes The ideal probe length is debatable but 200 to 500 bases work
well. Longer probes may be more sensitive but can be difficult to
hybridize to their targets Radioisotopically labelled probes are very sensitive but yield
lower resolution with results being more difficult to assess Biotinylated probes may be problematic due to endogenous biotin
and biotin-binding proteins
NON ISOTOPIC IN SITU HYBRIDIZATION (NISH)
Like antibodies, each probe must be individually optimized for reactivity in tissues, with the
variables to consider including;
NON ISOTOPIC IN SITU HYBRIDIZATION (NISH)
Like antibodies, each probe must be individually optimized for reactivity in tissues, with the
variables to consider including;
Probe length Probe labelling Probe concentration Protease
concentration Hybridization
conditions Stringency washes Detection
methodology
Probe length Probe labelling Probe concentration Protease
concentration Hybridization
conditions Stringency washes Detection
methodologyBreast cancer peri-tumour angiogenesis. NISH using a digoxygenin-labelled VEGF riboprobe
BENEFITS OF IHC AND NISH ASSAYSBENEFITS OF IHC AND NISH ASSAYS
Specific, high resolution detection of targets in human tissue
Maintenance of tissue morphology
Histopathological identification
Identification of cell types
Comparison of normal and diseased tissue
Specific, high resolution detection of targets in human tissue
Maintenance of tissue morphology
Histopathological identification
Identification of cell types
Comparison of normal and diseased tissueBreast cancer peri-tumour angiogenesis.
NISH using a digoxygenin-labelled TGFbeta riboprobe localises to lymphocytes (Blue).
IHC using a APAAP and Fast Red and CD31 localises to endothelial cells (Red).
BENEFITS OF IHC AND NISH ASSAYSBENEFITS OF IHC AND NISH ASSAYS
In cases where a cell\type may not be recognised by morphology alone, multiple labelling studies can be used
The use of quality controlled reagents and techniques yields consistent and reproducible results
Paraffin-embedded specimens allow for retrospective studies
In cases where a cell\type may not be recognised by morphology alone, multiple labelling studies can be used
The use of quality controlled reagents and techniques yields consistent and reproducible results
Paraffin-embedded specimens allow for retrospective studies
Breast cancer peri-tumour angiogenesis.
NISH using a digoxygenin labelled TGFbeta riboprobe and APAAP/Fast Red localises to lymphocytes (Red).
IHC using immunoperoxidase and DAB CD31 localises to endothelial cells (Brown).
BENEFITS OF IHC AND NISH ASSAYSBENEFITS OF IHC AND NISH ASSAYS
Human tissue is more likely to reflect ‘real life’ expression of a target than cell lines
In ‘grind and find’ assays (such as western and northern blots) tissue architecture is lost and so is cell-specific resolution
A target expressed at high levels by cells which are present in small numbers in the tissue as a whole can be identified
Human tissue is more likely to reflect ‘real life’ expression of a target than cell lines
In ‘grind and find’ assays (such as western and northern blots) tissue architecture is lost and so is cell-specific resolution
A target expressed at high levels by cells which are present in small numbers in the tissue as a whole can be identified
Breast cancer peri-tumour angiogenesis.
NISH using a digoxygenin labelled TGFbeta riboprobe and APAAP/Fast Red localises to lymphocytes (Red).
Immunofluorescence with McAb CD31 localises to endothelial cells (Green). Endothelial cells expressing TGFbeta = Yellow.
Laser Capture Microdissection for Molecular AnalysisLaser Capture Microdissection for Molecular Analysis
Before AfterCapture
DNA RNA
cDNA microarraysDNA fingerprintingMutation analysis
Protein
Proteomics
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Pixcell II systemExpert pathologists
Transcription biologists
0.3 mm tissue cores
Immunohistochemistry
In Situ Hybridisation
TMA constructioneg. Breast cancerPathology input
Expression Profiles in Clinical Series: Tissue MicroarraysExpression Profiles in Clinical Series: Tissue Microarrays
Applications of Tissue Microarrays (TMAs)Applications of Tissue Microarrays (TMAs)
Characterisation of new antibodies for IHC
Gene expression profiling for differential diagnosis
Gene expression profiling for carcinoma of unknown primary site
Gene expression profiling for molecular subclassification of tumours
Array based comparative genomic hybridisation (ACGH) for differential diagnosis
Gene expression profiling and/or ACGH for identification of molecular therapeutic targets with the goal of achieving individualised therapy
Characterisation of new antibodies for IHC
Gene expression profiling for differential diagnosis
Gene expression profiling for carcinoma of unknown primary site
Gene expression profiling for molecular subclassification of tumours
Array based comparative genomic hybridisation (ACGH) for differential diagnosis
Gene expression profiling and/or ACGH for identification of molecular therapeutic targets with the goal of achieving individualised therapy
GENE ARRAYS
• one sample• many markers
• Gene expression
• Gene Amplification/deletion
TISSUE ARRAYS
many samples one marker
Antibodies
In situ hybridisation
The Future of Histopathology -The Concept of ‘Pathology IT’ and
Individualised Diseased ‘Tissue Profiling’
The Future of Histopathology -The Concept of ‘Pathology IT’ and
Individualised Diseased ‘Tissue Profiling’
Automated Histopathology, IHC, NISH and Image Analysis
Multiple IHC markers on one slide Combined IHC and in-situ RNA profiling In situ detection of multiple RNA transcription sites
(using NISH or FISH) Multivariate analysis of imaging and protein and mRNA
expression
Disease/tumour profiling for the individual patient with predictive and prognostic implications, predictive information regarding drug responses
Implications for future clinical trials work
Automated Histopathology, IHC, NISH and Image Analysis
Multiple IHC markers on one slide Combined IHC and in-situ RNA profiling In situ detection of multiple RNA transcription sites
(using NISH or FISH) Multivariate analysis of imaging and protein and mRNA
expression
Disease/tumour profiling for the individual patient with predictive and prognostic implications, predictive information regarding drug responses
Implications for future clinical trials work
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