THE ROLE OF THE PATHOLOGIST IN TARGETED THERAPY
& PERSONALIZED MEDICINE Dr Dinah Parums, Principal Pathologist, 2007
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 processing4) Tissue sectioning5) Archiving blocks and slides6) Data tracking / IT7) Histochemistry (ie. tinctorial stains such as H&E)8) Histopathology (microscopic morphological interpretation) *9) Image analysis / morphometry / microdissection10) Antibody acquisition (internal or commercially) *11) Antibody validation (westerns + IHC + histopathology + controls) *12) Immunohistochemistry and immunofluorescence13) IHC quantitation14) Method development for IHC *15) Multiple IHC methods / multiplexing16) Confocal microscopy17) Electronmicroscopy18) Immunoelectronmicroscopy19) Non-isotopic in situ hybridisation for mRNA (NISH) / FISH20) Method development for NISH21) Combined IHC & NISH22) 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 MedicineHistopathology Capability
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.
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).
• 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
• 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 skinConfocal immunofluorescence
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 ?
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 methodologyBreast cancer peri-tumour angiogenesis.
NISH using a digoxygenin-labelled VEGF riboprobe
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).
Laser Capture Microdissection for Molecular Laser Capture Microdissection for Molecular AnalysisAnalysis
Before AfterCapture
DNA RNA
cDNA microarraysDNA fingerprintingMutation analysis
Protein
Proteomics
5 62
117 12910 138
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23
245 62
117 12910 138
314
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2455 6622
111177 1212991010 131388
331414
1144
1515
1616
17171818 1919
2020
2121
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2424
Pixcell II systemExpert pathologists
Transcription biologists
• 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)
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
HHPatient gets diagnosed &
tumour or blood sample taken
Patient is treated with eg. IRESSABiomarker 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.
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 ?
Tissue Reception Area
GLP
-80oCSecure Storage
GLP
Frozen Tissue
Room TempSecure Storage
GLP
FFPE Tissue
Histopath Lab
Tissue Repository
Human Tissue Microtome
GLP
Human Tissue Cryostat
GLP
Containment Level 2
Tissue Banking - Operating Model
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.
Biomarkers in Cancer
Pathogenesis
Risk Assessment
Early Detection
Prognostic Markers
New Therapies
Chemoprevention
Basic and Translational Research
Biomarkers Development
PathologyDiagnosis
Tissue BankBiomarkers
BIOMARKERS IN CANCER eg. LUNG CANCER PATHOLOGY
Squamous Cell Carcinomal
SCLCSmall Cell Carcinomal
Adenocarcinoma
Bronchioloalveolar cell carcinoma (BALC)
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
SquamousDysplasia
BronchusAngiogenic Squamous Dysplasia
Bronchus/Bronchiole
InflammatoryChanges
Small Bronchus/Bronchiole
NormalEpithelium
AlveoliAdenomatous
AlveolarHyperplasia
Small Bronchus/Bronchiole
NormalEpithelium
Adenocarcinoma
Small Cell Carcinoma
BronchusNormal
Epithelium/Hyperplasia
MycTP53
Genetic Instability
TSGs-Chr 3p 9p (p16)
MethylationAkt-mTOR
AngiogenesisVEGF/VEGFR
NF-BCOX-2
Angiogenesis
Unknown
KRAS Signaling p16 - LKB1
EGFR Signaling
Wistuba, 2006
Multiple Marker Analysis in Lung Cancer Tissue Specimens
Epidermal Growth Factor Receptor (EGFR)
Proliferation
Invasion MetastasisAngiogenesis
Resistance toapoptosis
Cell membrane
Ligand: EGF, TGF-a, AR
Nucleus
Gene transcriptioncell-cycle progression
ATP ATPPI3K
Akt STAT MAPK MEK
EGFR-TK
RAF
RASSOS
GRB2P
EGFR-TKpathways
Dec/01Dec/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
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)
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).
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.
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.
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.
‘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
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
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
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
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
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
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
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
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
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.
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
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
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
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
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.
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