Dr Dinah Parums. The Role of The Pathologist in Target Identification and Validation in Targeted Therapy and Personalized Medicine.

1. THE ROLE OF THE PATHOLOGIST IN
DRUG DISCOVERY AND DEVELOPMENT
- TARGET VALIDATION
Dr 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.

2. 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

3. WHAT CAN Immunohistochemistry (IHC)
SHOW ? ?
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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 skin
Confocal immunofluorescence

4. WHAT CAN IHC (and
IMMUNOFLUORESCENCE) SHOW ?
Double confocal
immunofluorescence
McAb CD31
localizing
to endothelial cells
(Red)
McAb ASMA
localizing
to smooth muscle cells
(Green)

5. CONSIDERATIONS 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
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. 6 KEY STEPS FOR IHC
IN TUMOUR BIOMARKER DEVELOPMENT
1 NEED FOR SPECIFIC ANTIBODY
TO IDENTIFY PROTEIN MARKER.
Custom made
Commercially available
Academic institution
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 formalinfixed).
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.

7. NON ISOTOPIC IN SITU HYBRIDIZATION
(NISH) STUDIES
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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

8. NON ISOTOPIC IN SITU HYBRIDIZATION
(NISH)
Like antibodies, each probe must be individually
optimized for reactivity in tissues, with the
variables to consider including;
Breast cancer peri-tumour angiogenesis. NISH
using a digoxygenin-labelled VEGF riboprobe
 Probe length
 Probe labelling
 Probe
concentration
 Protease
concentration
 Hybridization
conditions
 Stringency washes
 Detection
methodology

9. BENEFITS 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
Breast cancer peri-tumour angiogenesis.
NISH using a digoxygenin-labelled TGFbeta riboprobe normal and diseased
tissue
localises to lymphocytes (Blue).
IHC using a APAAP and Fast Red and CD31 localises
to endothelial cells (Red).

10. BENEFITS OF IHC AND NISH ASSAYS
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).
 In cases where a
celltype 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

11. BENEFITS 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
Breast cancer peri-tumour angiogenesis.
cell-specific
NISH using a digoxygenin labelled TGFbeta riboprobe
resolution
and APAAP/Fast Red localises to lymphocytes (Red).
 A target expressed
Immunofluorescence with McAb CD31 localises to
at high levels by
endothelial cells (Green). Endothelial cells expressing
cells which are
TGFbeta = Yellow.
present in small

12. Laser Capture Microdissection for Molecular Analysis
Pixcell II system
Expert pathologists
Transcription biologists
Before
DNA
Mutation
analysis
DNA fingerprinting
Capture
After
RNA
cDNA microarrays
Protein
Proteomics
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13. Expression Profiles in Clinical Series: Tissue Microarrays
0.3 mm tissue
cores
Immunohistochemistry
TMA construction
eg. Breast cancer
Pathology input
In Situ Hybridisation

14. Applications of Tissue Microarrays
(TMAs)
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Characterisation of new
antibodies for IHC
Gene expression
profiling for
differential diagnosis
Gene expression
profiling for carcinoma
of unknow n 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
GENE ARRAYS
TISSUE ARRAYS
•
•
one sample
many markers
many samples
one marker
•
Gene
expression
Antibodies
•
Gene
Amplification/d
eletion
In situ hybridisation

15. The Future of Histopathology The Concept of ‘Pathology IT’ and
Individualised Diseased ‘Tissue Profiling’
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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