dr nik zeps sir charles gairdner hospital
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Apoptosis and Prediction of Response to Chemotherapy in Breast Cancer: The Role of Molecular Markers and Digital Imaging in Clinical Practice. Dr Nik Zeps Sir Charles Gairdner Hospital. Breast Cancer. Breast Cancer affects 1 in 8 Australian women 1 Greatest number of cancer-related deaths 1 - PowerPoint PPT PresentationTRANSCRIPT
Dr Nik Zeps
Sir Charles Gairdner Hospital
Apoptosis and Prediction of Response to Chemotherapy in Breast Cancer: The Role of
Molecular Markers and Digital Imaging in Clinical Practice
Breast Cancer
• Breast Cancer affects 1 in 8 Australian women1
• Greatest number of cancer-related deaths1
• Divergence in patient outcome
• Cannot accurately predict patient outcome or disease course
1. AIHW Breast cancer in Australia: an overview, 2006
Polychemotherapy for early breast cancer: an overview of the randomised trials
Early Breast Cancer Trialists' Collaborative Group*Lancet 1998 Volume 352 Issue 9132 Page 930-942
Interpretation•< 3 women with good prognosis derive gain per 100 treated•c. 7 women with node -ve derive gain per 100 treated• c. 11women with node -ve derive gain per 100 treated
•NB these women are all pre or peri-menopausal:•Chemo could be simply causingchemical ovariectomy!•Could just give Tamoxifen/LHRH agonists
Polychemotherapy for early breast cancer: an overview of the randomised trials
Early Breast Cancer Trialists' Collaborative Group*Lancet 1998 Volume 352 Issue 9132 Page 930-942
Interpretation•< 1 woman with good prognosis derives gain per 100 treated•< 3 women with node -ve derive gain per 100 treated•< 4 women with node -ve derive gain per 100 treated
•NB these women are all postmenopausal:•Chemo really has no benefit at all
Chemotherapy in Breast Cancer
• Where is the evidence for treating EVERY woman?
• Only marginal benefit for treating 100% of women
• Imperative to discriminate which women will get benefit– avoid over-treating >90% of women who don’t
need it
Personalised Medicine
• Individualised therapy based on clinical and molecular markers
• Requires – Biomarker
discovery– Validation
While the other explosives played during recess, Charlie studied diligently in hopes of one day
becoming a Smart Bomb
http://www.junkertoons.com/toonbomb.htm
those placed in the low-risk category by the microarray analysis had a 96% probability of overall survival and 87% likelihood of disease freesurvival for 10 years, whereas those with a high-risk profile had a 50% overall survival and 48% probability of disease-free survival for 10 years.
MINDACT (Microarray In Node negative Disease may Avoid ChemoTherapy)
http://www.eortc.be/services/unit/mindact/MINDACT_websiteii.asp
How do you do this?
• Need Samples– Biobanks
• Quality material• Clinical Annotation
• Issues– Where are they?– How well set up are they?– Do they have enough samples?– Are samples processed suitably– Clinical information?
• Treatment• Follow Up
“The pathologists, none of whom in the lead cases were responsible for obtaining consent for post-mortems, bear no responsibility for the failure which I have found to exist.”
The Hon Mr Justice Gage
Trust: Comes on foot, leaves on horseback
"The crowd of foolish scribblers is scarcely less than the swarms of flies in the height of summer, and threatens with their crude and flimsy productions to stifle us as with smoke." William Harvey
Knock on effect of Alderhey is a general mistrust of doctors’ intent with human tissue
RPH
Mount
KEMH
Joondalup
SJOG
SCGH
SJOG
Fremantle
10km
•Carries out
–Consent
–Collection
–Processing
–Storage
–Data linkage (WAGER)
•AOCS (ovarian)-1000 (200)
•RADAR (prostate) -400
•IAB (Prostate)-150
•TARGIT IORT (Breast)-200
•Cancer Fatigue 100
•LABC 50 pa
•Colorectal 400 pa
•Sporadic breast 300 pa
WA Oncology Collection
Clinical Annotation
• Clinical Trial – Standardised therapy– Randomisation– Controls– Rich detail
• Multidisciplinary Group– As above except not randomised
Locally Advanced Breast Cancer (LABC)
• 10% of breast cancers • Large tumours, skin involvement, invasion to axillary lymph nodes,
inflammatory
• Management: neoadjuvant chemotherapy, surgery, adjuvant therapy (radiotherapy, chemotherapy, hormonal therapy)
• In vivo model of response to therapy
Locally advanced breast Cancer
• Neoadjuvant therapy (TAC)• T3 or T4, or N2 or N3 and M0 breast cancer • TAC (Docetaxel 75mg/m2, Doxorubicin
50mg/m2, Cyclophosphamide 500mg/m2 given d1 q 3 weeks
• Pts w clinical SD/PR or CR after 6 cycles TAC, – breast conserving surgery based on
• patient preference, residual tumour size < 4cm, acceptable cosmesis,
• absence of extensive microcalcification, absence of multifocal disease, non-inflammatory cancer and ability to achieve minimal of 5mm margin clearance
LABC tissue collection
• Multi-centre collection– Mount, RPH, SCGH
• Collected in parallel with clinical trial
• 44 patients gave consent
Apoptosis & Predictive Markers
• Chemotherapy induces apoptosis
• Many chemo agents induce cell death through DNA damage
• Many chemo agents induce mitochondrial-dependent apoptosis
αβ β
γ
δ
a• Cytochrome C release
• Acidification of cytosol early event in apoptosis
• pH regulated by ATP synthase
• Apoptotic stimuli causes reverse operation of the ATP synthase pump
ATP synthase & apoptosis
Cytosol
IMS
Mt matrix
ββ
εγ
δ
α
a
APOPTOSIS
BAX
BAX
αβ β
γ
δ
a• ATP synthase blockers inhibited Bax-induced cell death and cyt C release in yeast models.
• Restoration of mutated ATP synthase subunits restored sensitivity to Bax
• Does not completely abrogate apoptotic signaling – affects efficiency
How important is the ATP synthase pump in apoptosis?
Cytosol
IMS
Mt matrix
β-F1-ATP synthase and its role in predicting
response to chemotherapy in locally advanced breast cancer
• Otto Warburg 1931 Nobel prize– Glycolysis higher in cancer cells
• β-F1-ATP synthase rate limiting step for making ATP
• Bax can induce apoptosis– requires functional ATP synthase to do so– loss of the proton pump may prevent Bax from
integrating into the outer mitochondrial membrane to form a channel and facilitate cytochrome c release
Hypothesis
Patients with impaired mitochondrial function – Mitochondrial number– Mitochondrial DNA mutations– ATPase expression
will have poorer response rates to chemotherapy than patients with normal mitochondrial function
Scientific Approach
• Immunohistochemistry – Specific mitochondrial proteins
• PCR– Mitochondrial number– Integrity of mitochondrial DNA
Tissue Micro-arrays
• 500 patients 10 slides
• 400 with normal tissue
• Clinico-pathological features of all cases
• Treatment and survival data
Results to date
• Staining breast TMAs complete
• Collaboration established with Molecular Discovery Systems for staining analysis
• β software– Volume– Image size– Data set size
• 6.6GB Xcel Data from 19 TMA cores
4x
40x40x
Results to date: image de-convolution and visualisation
Automated cell finding.
Original Nucleus Nuclei detected Erosion filter
Nuclei touchingSome nuclei still clumped
Nuclei de-clumped
However, if you require the nuclei to be fully separated the segmentation can be reduced to a seed , this is of particular use if the cytoplasm is what is required for measurements and nucleus just for identification of object.
IHC
How refined is your endpoint?
Switch from traditional assay output to digital output.
Refine the Map for Higher Magnification
10X = 750 tiles 20X = 3000 tiles 40X = 15000 tiles
Output Changes
Issues
• Most systems have proprietary file formats eg Aperio .svs
• The images of full slides can be 13GB
• Compression of images as a way of controlling image size leads to loss of information and image artefact
Analysis Measurements, which ones, and how many??
Link to Health information
Summary
• Need samples for marker development
• Have to be good quality– Good clinical annotation
• Need projects
• Image analysis complex
• Must be linked to clinical info for outcomes
Acknowledgements
Sally Mclaren, PhD student
Technical • Sam Gallagher (MDS)• Lisa Spalding• Anne-Marie Shearwood
Funding• Lions Cancer Institute• National Health and Medical Research Council• Radiation Oncology, SCGH• University of Western Australia• Cancer Council of WA
Tissue Collection• SCGH breast clinic/ RPH breast assessment clinic (Prof Christobel Saunders)• Mount Hospital breast surgeons: Dr Hastrich, Mr Ingram, Mr Wilsher• Dr Arlene Chan• PathWest • Western Diagnostic Pathology