Dr Dinah Parums. The Role of the Pathologist in Targeted Therapy and Personalized Medicine (NSCLC)

transcript

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

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1718 19

245 62

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2455 6622

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331414

17171818 1919

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

•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

-80oCSecure Storage

Frozen Tissue

Room TempSecure Storage

FFPE Tissue

Histopath Lab

Tissue Repository

Human Tissue Microtome

Human Tissue Cryostat

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

RASSOS

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

Sample Limitations

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

Sample Limitations

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

Sample Limitations

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.

Dr Dinah Parums. The Role of the Pathologist in Targeted Therapy and Personalized Medicine (NSCLC)

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