The Role of the Pathologist in Targeted Therapy and Personalized Medicine in NSCLC

1. THE ROLE OF THE PATHOLOGIST
IN TARGETED THERAPY
& PERSONALIZED MEDICINE
Dr Dinah Parums, Principal Pathologist, 2007

2. WHAT ARE THE COMPONENTS OF A PATHOLOGY
CAPABILITY ?
1) Tissue acquisition (clinical, commercial,CPU, external trial material) *
2) Tissue fixation and/or storage *
3) Tissue processing
4) Tissue sectioning
5) Archiving blocks and slides
6) Data tracking / IT
7) Histochemistry (ie. tinctorial stains such as H&E)
8) Histopathology (microscopic morphological interpretation) *
9) Image analysis / morphometry / microdissection
10) Antibody acquisition (internal or commercially) *
11) Antibody validation (westerns + IHC + histopathology + controls) *
12) Immunohistochemistry and immunofluorescence
13) IHC quantitation
14) Method development for IHC *
15) Multiple IHC methods / multiplexing
16) Confocal microscopy
17) Electronmicroscopy
18) Immunoelectronmicroscopy
19) Non-isotopic in situ hybridisation for mRNA (NISH) / FISH
20) Method development for NISH
21) Combined IHC & NISH
22) In situ PCR
* Critical Components of
Discovery Medicine IHC Group
Components of Molecular
Pathology (in development)
Components of a
Tissue Bank
Methods in development
or done externally
Discovery Medicine
Histopathology
Capability

3. Challenges for the Pathologist in
Drug Discovery
• Antibody characterisation;
• Standardization of IHC techniques;
• IHC method quality control;
• Management of workflow;
• Analysis and interpretation of IHC data;
• Archiving of IHC image data.

4. THE ROLE OF THE PATHOLOGIST IN DRUG
DISCOVERY AND DEVELOPMENT
- TARGET VALIDATION
• 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) pinpointing the expression of protein (combination
with NISH can also show nucleic acids).

5. • 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
IMMUNOHISTOCHEMISTRY and
IMMUNOFLUORESCENCE

6. • 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)
WHAT CAN IMMUNOHISTOCHEMISTRY AND
IMMUNOFLUORESCENCE SHOW ?
McAb ASMA in myofibroblasts in healing skin
McAb ASMA in myofibroblasts in healing skin
Confocal immunofluorescence

7. 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 ?

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;
• Probe length
• Probe labelling
• Probe concentration
• Protease concentration
• Hybridization conditions
• Stringency washes
• Detection methodology
Breast cancer peri-tumour angiogenesis.
NISH using a digoxygenin-labelled VEGF
riboprobe

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 normal
and diseased tissue
Breast 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).

10. Laser Capture Microdissection for MolecularLaser Capture Microdissection for Molecular
AnalysisAnalysis
Before AfterCapture
DNA RNA
cDNA microarraysDNA fingerprintingMutation
analysis
Protein
Proteomics
5 6
2
11
7
129
10 138
3
14
1
4
15
16
17
18 19
20
21
22
23
24
5 6
2
11
7 12
9
10 138
3
14
1
4
15
16
17
18 19
20
21
22
23
24
55 66
22
1111
77 121299
1010 131388
33
1414
11
44
1515
1616
1717
1818 1919
2020
2121
2222
2323
2424
Pixcell II system
Expert pathologists
Transcription biologists

11. • 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
Applications of Tissue Microarrays (TMAs)

12. The Future of Pathology
‘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

13. HH
Patient gets diagnosed &
tumour or blood
sample taken
Patient is treated
with eg. IRESSA
Biomarker analysis
Patient benefits
The development of predictive Companion Diagnostic
Biomarkers accompany molecularly targeted therapies in
clinical practice.
Test is positive
PERSONALISED MEDICINE
those patients that have a particular biomarker will benefit.

14. EXISTING TARGETED THERAPIES WITH
COMPANION DIAGNOSTICS
•Tamoxifen
–ER
•Trastuzamab (Herceptin)
–ErbB2 amplification – Breast cancer
•Imatinib (Glivec)
–Bcr-abl translocation - CML
•Imatinib (Glivec)
–c-KIT IHC - GIST
•Erlotinib (Tarceva)/Gefitinib (Iressa)
–EGFR IHC/ISH ?

15. Tissue Reception
Area
GLP
-80o
C
Secure Storage
GLP
Frozen Tissue
Room Temp
Secure Storage
GLP
FFPE Tissue
Histopath Lab
Tissue Repository
Human Tissue
Microtome
GLP
Human Tissue
Cryostat
GLP
Containment Level 2
Tissue Banking - Operating Model

16. Challenges for the Pathologist in
Drug Development;
Tissue Biomarkers in Clinical Trials
• Implementation of tissue sampling while
managing the impact on patient enrollment, cost
and sample disposition;
• Development of best practices and processes for
standardization of tissue collection to minimize
the effect of pre-analytical variables on
downstream results;
• Balancing ‘intellectual’, hypothesis-seeking
approaches with practical, cost-effective assays
that can be performed on individual patients.

17. Biomarkers in Cancer
Pathogenesis
Risk Assessment
Early Detection
Prognostic Markers
New Therapies
Chemoprevention
Basic and Translational Research
Biomarkers Development
Pathology
Diagnosis
Tissue Bank
Biomarkers

18. BIOMARKERS IN CANCER eg. LUNG CANCER
PATHOLOGY
Squamous Cell Carcinomal
SCLCSmall Cell Carcinomal
Adenocarcinoma
Bronchioloalveolar cell
carcinoma (BALC)

19. Multiple Histopathologic and Molecular Pathways in Lung Cancer Pathogenesis
Clinical Features
Squamous Cell
Carcinoma
Bronchus
Pathologic Changes Molecular Changes
Smoking
(with or without COPD)
Non-Smoking
Squamous
Dysplasia
Bronchus
Angiogenic
Squamous
Dysplasia
Bronchus/
Bronchiole
Inflammatory
Changes
Small
Bronchus/
Bronchiole
Normal
Epithelium
Alveoli
Adenomatous
Alveolar
Hyperplasia
Small
Bronchus/
Bronchiole
Normal
Epithelium
Adenocarcinoma
Small Cell Carcinoma
Bronchus
Normal
Epithelium/
Hyperplasia
Myc
TP53
Genetic
Instability
TSGs-Chr 3p
9p (p16)
Methylation
Akt-mTOR
Angiogenesis
VEGF/VEGFR
NF-κB
COX-2
Angiogenesis
Unknown
KRAS Signaling
p16 - LKB1
EGFR Signaling
Wistuba, 2006

20. Multiple Marker Analysis in Lung Cancer Tissue
Specimens

21. Epidermal Growth Factor Receptor
(EGFR)
Proliferation
Invasion Metastasis
Angiogenesis
Resistance to
apoptosis
Cell membrane
Ligand: EGF, TGF-a, AR
Nucleus
Gene transcription
cell-cycle progression
ATP
ATPPI3K
Akt
STAT MAPK MEK
EGFR-TK
RAF
RAS
SOS
GRB2
P
EGFR-TK
pathways

22. Dec/01De
c/01
Dec/00
EGFR Mutations and TK Inhibitors in Lung Cancer
Activating EGFR Mutations in Lung
Cancer Correlate with Clinical Response to
EGFR Inhibitors (Paez et al, Science and
Lynch et al, NEJM, April-May 04)
Groups with High Frequency of
Mutations:
• Adenocarcinoma
• Women
• Non-smokers
• People of Asian Descent

23. IRESSA SURVIVAL EVALUATION IN LUNG
CANCER (ISEL) TRIAL – STUDY 709
• Phase III trial comparing gefitinib with placebo in
1,692 patients with refractory advanced NSCLC
• Biomarkers
– EGFR IHC (n=379)
– EGFR FISH (n=370)
– P-Akt expression (n=382)
– Mutations in EGFR (n=215), KRAS (n=152), BRAF
(n=118)

24. ISEL
702 TISSUE SAMPLES REVIEWED IN 2004/2005
• A total of 702 cases have been examined out of which 552 (78.6%) of cases arrived as
blocks and 122 (17.4%) of cases as slides only.
• 11 cases were tissue scrapes in eppendorfs with no slides or blocks; 7 cases were single
stained slides with no extra sections or blocks.
• Out of these 702 cases, 192 (27.4%) were inadequate either because there was no tissue or
there was no tumour or else because the tissue was so poorly fixed that the morphology
could not be interpreted.
• Out of the total 510 adequate cases, all proceeded to DNA extraction from marked thick or
thin sections and to IHC for EGFR.
• Out of the adequate cases with tissue blocks, there were 144 (20%) resection cases with
sufficient tumour in the blocks for extra sections (deemed as non biopsy material with
tumour present > 5 mm in any dimension).

25. HISTOPATHOLOGY REPORT DATA FIELDS FOR ISEL
• E number.
• DM number or study case number (anonymised).
• Specimen (Biopsy or Resection).
• Tissue (Lung, Bronchus, Pleura, indeterminate).
• Adequate Tissue (Yes/No).
• Adequate Fixation (Yes/No).
• Diagnosis (NSCC – non small cell carcinoma; NSCT – non small cell tumour; OT – other tumour;
NT – no tumour).
• IEN – intra-epithelial neoplasia (Yes/No).
• Greatest dimensions of tissue (xmm x ymm) (measured on the slide using the microscope Vernier).
• Greatest dimensions of tumour(a mm x b mm).
• Inflammation (as a % of the tumour area).
• Necrosis (as a % of the tumour area).
• Mitosis (% cells as measured at x20 objective).
• Apoptosis (% cells as measured at x20 objective).
• COMMENT – add reasons for inadequacy, qualify diagnosis with SCC or adenocarcinoma etc.

26. Immunohistochemistry for EGFR using the DAKO IHC
kit and automated immunostaining
 Automated immunostaining
methods for EGFR ensure
reproducibility.
The DAKO PharmDxTM
kit is
designed for automated
immunohistochemistry and
slides can be batched.

27. Immunohistochemistry for EGFR using the DAKO IHC
kit and automated immunostaining
(brown staining of cell membrane)
 non small cell carcinoma
 objective x 20
 95% of tumour cells are
positive
 80% are 3+
 10% are 2+
 5% are 1+
 and 5% are O.

28. ‘By Eye’ Quantitation of EGFR IHC
(brown staining of cell membrane)
The ‘H’ Score
percentage +ve H score
0 1+ 2+ 3+
0 0 0 100 (1x1+) + (2x2+) + (3x3+)
maximum = 300
Example case
5 5 10 80 H score = 265

29. SUMMARY REQUIREMENTS FOR TISSUE
SUBMISSION FOR PATHOLOGY INVESTIGATORS
FOR PHASE III CLINICAL TRIALS
• The patient should have available a primary diagnostic tumour biopsy, obtained prior to
treatment, if possible.
• Tissue must be adequately fixed in 10% neutral buffered formalin (we can provide a protocol).
• Tissue must be embedded in paraffin wax and in a plastic cassette with clear identification.
• The histopathology of the tissue remaining in the block must be QC’d by a site Histopathologist
to confirm the tissue identification and the tumour diagnosis.
• We require adequately fixed tissue with good cell morphology.
• We require adequate amounts of tumour present remaining in the block,
(> 100 tumour cells) eg.
• Good cell morphology
• >100 cells
• Non small cell carcinoma
• Ideally, we would wish to be sent the QC’d tissue block.
• If it is not possible to send us the tissue block, then we wish to receive NO LESS THAN 16
unstained sections, cut at 5 micron thickness on to clean ‘SuperFrost’glass slides and with a
new disposable microtome blade used for each patient

30. CHALLENGES FOR PATHOLOGISTS IN CLINICAL
BIOMARKER DEVELOPMENT
LOGISTIC Sample Collection
TECHNICAL Pre-Analytical Variables
Primary Antibody Selection
Sample Limitations
CONCEPTUAL Primary vs Metastasis
Single vs Multiple Biomarkers

31. CHALLENGES FOR PATHOLOGISTS IN CLINICAL
BIOMARKER DEVELOPMENT
LOGISTIC Sample Collection
TECHNICAL Pre-Analytical Variables
Primary Antibody Selection
Sample Limitations
CONCEPTUAL Primary vs Metastasis
Single vs Multiple Biomarkers

32. LOGISTIC: WHY IS TISSUE COLLECTION SO
DIFFICULT IN THE CONTEXT OF A CLINICAL
TRIAL ?
• Inclusion of sample collection in clinical trial design
– Increases logistic complexity
– Potential IRB issues
– Has the potential to slow enrollment
– Increases cost
• Prospective biopsies
– Give most control over pre-analytical variables
– Adds the most logistic complexity and cost
– There is limited tissue
• Archival paraffin blocks
– These are relatively easy to collect
– There is no control over pre-analytical variables

33. CHALLENGES FOR PATHOLOGISTS IN CLINICAL
BIOMARKER DEVELOPMENT
LOGISTIC Sample Collection
TECHNICAL Pre-Analytical Variables
Primary Antibody Selection
Sample Limitations
CONCEPTUAL Primary vs Metastasis
Single vs Multiple Biomarkers

34. TECHNICAL : CONTROLLING PRE-ANALYTICAL
VARIABLES AND MINIMIZING VARIABILITY IN
DOWNSTREAM DATA
• Time to Fixation
• Time of Fixation
• Type of Fixation
• Use of phosphatase inhibitors
• Tissue processing protocol
• Embedding: paraffin temperature
• Type of glass slides (eg. Superfrost plus)
• Adequacy of deparaffinization
• Age of cut sections at time of analysis

35. TECHNICAL : POSSIBLE ALTERNATIVE SAMPLE
COLLECTION STRATEGIES
• Fine needle aspiration (FNA) samples
– Less invasive
– Sampling can be done more easily
– Yield can be high depending on expertise
– Limited sample quantity vs. core biopsy
• Circulating tumour cells
– Data suggests utility as a prognostic marker
– Unknown whether isolated cells are a valid surrogate for use in
biomarker studies
• Cancer stem cells

36. CHALLENGES FOR PATHOLOGISTS IN CLINICAL
BIOMARKER DEVELOPMENT
LOGISTIC Sample Collection
TECHNICAL Pre-Analytical Variables
Primary Antibody Selection
Sample Limitations
CONCEPTUAL Primary vs Metastasis
Single vs Multiple Biomarkers

37. CONCEPTUAL : DOES THE BIOMARKER READOUT
FROM THE PRIMARY TUMOUR ACCURATELY
REFLECT METASTATIC DISEASE ?
• Primary Tumour
– Basis for diagnosis
– Paraffin embedded archival tumour samples available
– Usual sample used for biomarker assessment
• Metastatic Tumour
– Target for investigational therapy
– Tissue sample less often available
– Additional biopsy may be required
eg.
Comparison of the epidermal growth factor receptor gene and protein in primary non small cell lung cancer and
metastatic sites: implications for treatment with EGFR inhibitors.
Italiano, A, Burel Vandenbos, F, Otto, J. et al. Annals of Oncology 17:2006;981-985.

38. CONCEPTUAL : Implications for Clinical Trials
• Assessment of putative predictive biomarkers need to be
done with knowledge of whether the primary or metastatic
sample was obtained and analyzed
• All samples collected in clinical trials need to be annotated
with anatomic site and identity: - ‘primary’ or ‘metastasis’
• Ideally, both the primary tumour and the metastatic sample
should be collected and analyzed

39. CONCEPTUAL : CAN A SINGLE BIOMARKER
ACCURATELY PREDICT CLINICAL OR
THERAPEUTIC OUTCOME ?
Molecular Profiling and Personalized Predictive Pathology
– ? Will this ever replace morphological assessment by the
Pathologist
• No – it is a natural extension of the work of Pathologists
– ? Part of the routine assessment of tumours by diagnostic
Pathologists
– ? Subspecialty labs
• ? Academic
• ? Commercial

40. OPPORTUNITIES FOR PATHOLOGISTS IN FUTURE
CLINICAL TRIALS
• Establish guidelines and best practices for sample
collection and preparation for predictive
biomarker development
– Time to fixation, time of fixation, cut slide oxygen
exposure
– Sample annotation including ‘primary’ vs ‘metastasis’
• Multiple antibody clones
• Examination of different scoring parameters and
cut-offs with outcome correlation
• Generation of drug-treated sample repository
– Interrogation of exploratory markers, profiles and
technologies

41. OPPORTUNITIES FOR PATHOLOGISTS IN THE ERA
OF PERSONALIZED MEDICINE
• Tissue Acquisition and Processing
– Approach to sample procurement
– Control of pre-analytical variables
• Assay Development
– Selection of primary antibody
– Selection of appropriate positive and negative controls
– Reduction of complex data sets and methods in
practical assays
• Design of Clinical and Companion Diagnostic
Studies
– Biomarker strategy and concept
– Data scoring methods/bioinformatics

42. Challenges for the Pathologist in
Drug Development;
Tissue Biomarkers in Clinical Trials
• Implementation of tissue sampling while
managing the impact on patient enrollment, cost
and sample disposition;
• Development of best practices and processes for
standardization of tissue collection to minimize
the effect of pre-analytical variables on
downstream results;
• Balancing ‘intellectual’, hypothesis-seeking
approaches with practical, cost-effective assays
that can be performed on individual patients.