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 materi...
Challenges for the Pathologist in
Drug Discovery
• Antibody characterisation;
• Standardization of IHC techniques;
• IHC m...
THE ROLE OF THE PATHOLOGIST IN DRUG
DISCOVERY AND DEVELOPMENT
- TARGET VALIDATION
• Biotechnology and pharmaceutical compa...
• Tissue sections from normal and
diseased specimens on glass
slides as whole sections,
multiblocks or TMAs
• Tissues are ...
• The detection of target
antigens (usually proteins)
within tissues and cells
• Relative level of target
expression
• Sub...
CONSIDERATIONS FOR ANTIBODY USE
• ‘Clean’ monoclonal and polyclonal antibodies should be used (confirmed by
western blot o...
NON ISOTOPIC IN SITU HYBRIDIZATION
(NISH)
Like antibodies, each probe must be individually optimized for
reactivity in tis...
BENEFITS OF IHC AND NISH ASSAYS
• Specific, high resolution
detection of targets in
human tissue
• Maintenance of tissue
m...
Laser Capture Microdissection for MolecularLaser Capture Microdissection for Molecular
AnalysisAnalysis
Before AfterCaptur...
• Characterisation of new antibodies
for IHC
• Gene expression profiling for
differential diagnosis
• Gene expression prof...
The Future of Pathology
‘Pathology IT’ and Individualised
Diseased ‘Tissue Profiling’
• Automated Histopathology, IHC, NIS...
HH
Patient gets diagnosed &
tumour or blood
sample taken
Patient is treated
with eg. IRESSA
Biomarker analysis
Patient ben...
EXISTING TARGETED THERAPIES WITH
COMPANION DIAGNOSTICS
•Tamoxifen
–ER
•Trastuzamab (Herceptin)
–ErbB2 amplification – Brea...
Tissue Reception
Area
GLP
-80o
C
Secure Storage
GLP
Frozen Tissue
Room Temp
Secure Storage
GLP
FFPE Tissue
Histopath Lab
T...
Challenges for the Pathologist in
Drug Development;
Tissue Biomarkers in Clinical Trials
• Implementation of tissue sampli...
Biomarkers in Cancer
Pathogenesis
Risk Assessment
Early Detection
Prognostic Markers
New Therapies
Chemoprevention
Basic a...
BIOMARKERS IN CANCER eg. LUNG CANCER
PATHOLOGY
Squamous Cell Carcinomal
SCLCSmall Cell Carcinomal
Adenocarcinoma
Bronchiol...
Multiple Histopathologic and Molecular Pathways in Lung Cancer Pathogenesis
Clinical Features
Squamous Cell
Carcinoma
Bron...
Multiple Marker Analysis in Lung Cancer Tissue
Specimens
Epidermal Growth Factor Receptor
(EGFR)
Proliferation
Invasion Metastasis
Angiogenesis
Resistance to
apoptosis
Cell membra...
Dec/01De
c/01
Dec/00
EGFR Mutations and TK Inhibitors in Lung Cancer
Activating EGFR Mutations in Lung
Cancer Correlate wi...
IRESSA SURVIVAL EVALUATION IN LUNG
CANCER (ISEL) TRIAL – STUDY 709
• Phase III trial comparing gefitinib with placebo in
1...
ISEL
702 TISSUE SAMPLES REVIEWED IN 2004/2005
• A total of 702 cases have been examined out of which 552 (78.6%) of cases ...
HISTOPATHOLOGY REPORT DATA FIELDS FOR ISEL
• E number.
• DM number or study case number (anonymised).
• Specimen (Biopsy o...
Immunohistochemistry for EGFR using the DAKO IHC
kit and automated immunostaining
 Automated immunostaining
methods for E...
Immunohistochemistry for EGFR using the DAKO IHC
kit and automated immunostaining
(brown staining of cell membrane)
 non ...
‘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 ...
SUMMARY REQUIREMENTS FOR TISSUE
SUBMISSION FOR PATHOLOGY INVESTIGATORS
FOR PHASE III CLINICAL TRIALS
• The patient should ...
CHALLENGES FOR PATHOLOGISTS IN CLINICAL
BIOMARKER DEVELOPMENT
LOGISTIC Sample Collection
TECHNICAL Pre-Analytical Variable...
CHALLENGES FOR PATHOLOGISTS IN CLINICAL
BIOMARKER DEVELOPMENT
LOGISTIC Sample Collection
TECHNICAL Pre-Analytical Variable...
LOGISTIC: WHY IS TISSUE COLLECTION SO
DIFFICULT IN THE CONTEXT OF A CLINICAL
TRIAL ?
• Inclusion of sample collection in c...
CHALLENGES FOR PATHOLOGISTS IN CLINICAL
BIOMARKER DEVELOPMENT
LOGISTIC Sample Collection
TECHNICAL Pre-Analytical Variable...
TECHNICAL : CONTROLLING PRE-ANALYTICAL
VARIABLES AND MINIMIZING VARIABILITY IN
DOWNSTREAM DATA
• Time to Fixation
• Time o...
TECHNICAL : POSSIBLE ALTERNATIVE SAMPLE
COLLECTION STRATEGIES
• Fine needle aspiration (FNA) samples
– Less invasive
– Sam...
CHALLENGES FOR PATHOLOGISTS IN CLINICAL
BIOMARKER DEVELOPMENT
LOGISTIC Sample Collection
TECHNICAL Pre-Analytical Variable...
CONCEPTUAL : DOES THE BIOMARKER READOUT
FROM THE PRIMARY TUMOUR ACCURATELY
REFLECT METASTATIC DISEASE ?
• Primary Tumour
–...
CONCEPTUAL : Implications for Clinical Trials
• Assessment of putative predictive biomarkers need to be
done with knowledg...
CONCEPTUAL : CAN A SINGLE BIOMARKER
ACCURATELY PREDICT CLINICAL OR
THERAPEUTIC OUTCOME ?
Molecular Profiling and Personali...
OPPORTUNITIES FOR PATHOLOGISTS IN FUTURE
CLINICAL TRIALS
• Establish guidelines and best practices for sample
collection a...
OPPORTUNITIES FOR PATHOLOGISTS IN THE ERA
OF PERSONALIZED MEDICINE
• Tissue Acquisition and Processing
– Approach to sampl...
Challenges for the Pathologist in
Drug Development;
Tissue Biomarkers in Clinical Trials
• Implementation of tissue sampli...
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Dr Dinah Parums. The Role of the Pathologist in Targeted Therapy and Personalized Medicine (NSCLC)

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

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  • Process is for Study Delivery to enter study level data onto DCMT & at tissue bank samples logged in & can be tracked. Michael Ripley & Sarah Rosamond were DCMT trained last week
    Scope of AZ Tissue Repository is All human tissue at AP/GHP except:
    Whole organs (no requirement), Xenograft tissue (stored separately), Commercially available cell lines (flow-through CIRA labs), Haematology samples (flow through SAUK/CRO labs), Clinical chemistry samples (flow through SAUK/CRO labs), Pharmacokinetic samples (flow through DMPK labs), PBMC DNA* (established RDG process), Protein Lysates
    Tissue sources (i) Commercial e.g. Asterand, Ventana (ii) NHS/academic centres e.g. Welsh Cancer Bank (CEG), Nottingham, Leeds, Bart’s/Royal London, Sheffield, Royal Marsden, Edinburgh (iii) legacy (iv) Clinical samples (v) DM Collaborations e.g. Liverpool, Oxford
    Capacity 12000 slides, 3000 FFPE, 4000 frozen
    no direct costs attributable to storage of human tissue samples within the central repository at Alderley Park
    extracted tissue and blood DNA will not be archived within the human tissue repository
    Specifically, the “ Cancer and Infection Human Tissue Repository" will not house non-human tissue, non-CIRA/CITA tissue
  • EPT1, 2, 5 & Iressa. EPT3 & Zactima in diary. EPTs interested in tailored biomarker session started off by EPT2 which Darren is co-ordinating
    Use MSP to bridge gap/demand in histopath workload spreadsheet
    Show Andrew Hughes’s Draft Paper for O&I TAPT
    Final draft of current CROs for IHC
    During the development of the CDP a number of tissue acquisition & IHC parameters need to be addressed with regard to the methodology for the collection, handling, storing of tissue, evaluation and presentation of IHC data. These parameters should be described in a strategic document to aide teams but not sure of differences & similarities of Pts to consider doc, TPD, Biomarker Plan?
    Bridge between SDT & Kerry for tissue bank samples arrive in appropriate tubes & labels, this week received urine (large bottles) & serum samples (sub-optimal labelling) from AP CPU in white plastic bags
  • Dr Dinah Parums. The Role of the Pathologist in Targeted Therapy and Personalized Medicine (NSCLC)

    1. 1. THE ROLE OF THE PATHOLOGIST IN TARGETED THERAPY & PERSONALIZED MEDICINE Dr Dinah Parums, Principal Pathologist, 2007
    2. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 18. BIOMARKERS IN CANCER eg. LUNG CANCER PATHOLOGY Squamous Cell Carcinomal SCLCSmall Cell Carcinomal Adenocarcinoma Bronchioloalveolar cell carcinoma (BALC)
    19. 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. 20. Multiple Marker Analysis in Lung Cancer Tissue Specimens
    21. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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.
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