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

5 62

117 12910 138

314

14

15

16

1718 19

20

21

22

23

245 62

117 12910 138

314

14

15

16

1718 19

20

21

22

23

2455 6622

111177 1212991010 131388

331414

1144

1515

1616

17171818 1919

2020

2121

2222

2323

2424

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