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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. 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

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Dr Nik Zeps Sir Charles Gairdner Hospital

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  1. 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

  2. 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

  3. Polychemotherapy for early breast cancer: an overview of the randomised trialsEarly 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 causing • chemical ovariectomy! • Could just give Tamoxifen/ • LHRH agonists

  4. Polychemotherapy for early breast cancer: an overview of the randomised trialsEarly 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 post • menopausal: • Chemo really has no benefit at all

  5. 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

  6. 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

  7. those placed in the low-risk category by the microarray analysis had a 96% probability of overall survival and 87% likelihood of disease free survival 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

  8. 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

  9. “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

  10. 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

  11. Joondalup 10km KEMH SJOG SCGH RPH Mount Fremantle SJOG WA Oncology Collection • 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

  12. Clinical Annotation • Clinical Trial • Standardised therapy • Randomisation • Controls • Rich detail • Multidisciplinary Group • As above except not randomised

  13. 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

  14. 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

  15. LABC tissue collection • Multi-centre collection • Mount, RPH, SCGH • Collected in parallel with clinical trial • 44 patients gave consent

  16. Apoptosis & Predictive Markers • Chemotherapy induces apoptosis • Many chemo agents induce cell death through DNA damage • Many chemo agents induce mitochondrial-dependent apoptosis

  17. δ a β β α γ α β β γ ε δ a ATP synthase & apoptosis Mt matrix • 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 IMS BAX Cytosol APOPTOSIS

  18. a γ α β β δ How important is the ATP synthase pump in apoptosis? Mt matrix • 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 IMS BAX Cytosol

  19. 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

  20. 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

  21. Scientific Approach • Immunohistochemistry • Specific mitochondrial proteins • PCR • Mitochondrial number • Integrity of mitochondrial DNA

  22. Tissue Micro-arrays • 500 patients  10 slides • 400 with normal tissue • Clinico-pathological features of all cases • Treatment and survival data

  23. 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 40x

  24. Results to date: image de-convolution and visualisation

  25. Original Nucleus Nuclei detected Erosion filter Automated cell finding. Nuclei de-clumped Some nuclei still clumped Nuclei touching 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.

  26. How refined is your endpoint? IHC Switch from traditional assay output to digital output.

  27. Refine the Map for Higher Magnification 10X = 750 tiles 20X = 3000 tiles 40X = 15000 tiles

  28. Output Changes

  29. 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

  30. Analysis Measurements, which ones, and how many??

  31. Link to Health information

  32. 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

  33. 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

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