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