1. TOP2A IS AN INDEPENDENT PREDICTOR OF SURVIVAL IN UNSELECTED BREAST CANCER Amit Pancholi Molecular Profiling of Breast Cancer: Predictive Markers of Long Term Survival and Tumour Classification

2. Outline Breast Cancer Background Classification Systems TOP2A Background Method Results Conclusion Future Work

3. Breast Cancer Background Each year in UK –41,700 women are diagnosed with breast cancer –Causes over 12,400 deaths Causes? –Certain drugs e.g. oral contraceptive pill and HRT –Genetic factors e.g. mutations of the tumour suppressor genes BRCA1 & BRCA2

4. Breast Cancer Morphology Mostly adenocarcinomas Breast Cancer InvasiveIn Situ Ductal – 80% Lobular – 10% Tubular – 1% Ductal – 5%Lobular – 6% Mucoid – 1% Medullary – 1%

5. Prognosis Ductal Carcinoma In Situ (DCIS) & Lobular Carcinoma In Situ (LCIS) – good prognosis –But may progress to invasive carcinoma Invasive carcinoma – ductal, lobular & mixed ductal-lobular– poor prognosis Uncommon specialised carcinomas – tubular, mucoid & medullary – good prognosis ( mets) Mixed ductal-specialised carcinomas – slightly higher prognosis

6. Prognostic Factors Prediction of survival –Tumour Stage (e.g. TMN Classification) Size, Lymph Nodes, Vascular Spread –Tumour grade Grade I III –Hormone receptor status Estrogen (ER) & Progesterone (PR): disease-free survival due to anti-hormone therapy

7. Nottingham Prognostic Index NPI = 0.2 x size (cm) + grade (1-3) + nodal stage (1-3) –Good prognostic group: GPG = NPI < 3.4 –Moderate: MPG = NPI 3.4 – 5.4 –Poor: PPG = NPI > 5.4

8. Molecular Factors Oncogene abnormalities –E.g. p53 HER-2 (erb-B2/neu) oncogene expression –Poorer prognosis

9. Current Classification Breast cancer subtypes devised by Perou et al. & Sorlie et al. –Luminal A. ER+ with good prognosis (survival>5yrs) –Luminal B. ER+ with poor prognosis (survival<5yrs) –Basal. ER-, PR-, Erb-B2- (Her-2/neu) –Erb-B2+. ER-, Erb-B2+ –Normal like. ER-, Erb-B2-, needs further classification.

10. Current Classification

11. Paik et al. were able to calculate a recurrence score which was validated as quantifying the probability of distant recurrence in tamoxifem-treated (ER antagonist), lymph node negative, ER+ breast cancer patients

12. Need for Further Classification Some breast cancers with the same histological type, tumour subtype and similar NPI scores have a very different outcome (survival) and response to treatment Need for a system that can be applied to a heterogeneous population of patients

13. Putative Marker Topoisomerase IIα gene (TOP2A) –Already important in breast cancer Encodes protein which is the molecular target for topoisomerase inhibitors such as anthracyclines in chemotherapy –Topoisomerase system mediates relaxation of the supercoiled double-stranded DNA helix for replication and transcription so there is no tension when it unwinds

14. Location TOP2A gene is located at 17q12-q21 on chromosome 17 –Close to Erb-B2 (Her-2/neu) oncogene, a widely accepted prognostic marker Many studies have shown that TOP2A is only amplified or deleted when Her-2 is amplified and TOP2A aberration with a normal copy number of Her-2 is unusual

15. Previous Studies Studies had suggested that TOP2A gene amplification was associated with poorer disease outcome in breast cancer –But it remained unclear whether its use as a prognostic marker was affected by its apparent dependence on Her- 2 amplification Nobody has reported any outcome & prognosis data for TOP2A deletion –However it is accepted that it is a cause of resistance to topoisomerase inhibitors We hypothesised that aberrations in the TOP2A gene will be associated with a poorer prognosis

16. FISH Fluorescent in situ hybridisation –2 fluorescently-labelled probes which had different DNA targets Green signal for the centromere of chromosome 17 Red signal for the TOP2A gene locus –Ratio of red:green signals determined aberration status E.g. >2 is amplification, <0.8 is deletion

17. CISH Colorimetric in situ hybridisation –Digoxigenin-labelled probe –>5 signals per cell in >50% of cancer cells is amplification

18. Tissue Microarrays Revolutionised cancer research Allows many patients to be screened in relatively short time We had 150 patient-tissue sections (cores) per slide

19. Results Results were analysed in a number of ways –Pearsons Chi-squared, Kaplan-Meier Survival curves, Cox Proportional Hazards Regression –Analysed for CISH, then FISH and CISH merged into one dataset –Uni- and multivariate analysis was used to test for association between TOP2A and clinical parameters Histological grade, distant metastases, tumour recurrence, tumour size, disease free interval & overall survival

21. Results CISH & FISH correlated significantly (Spearmans Rank p = 0.000) for the cases that were scored for both TOP2A was amplified in 25 cases out of 329 (7.6%) TOP2A amplification was significantly associated with high tumour grade (p<0.05), distant metastases (p<0.03) and tumour recurrence (p<0.015) No association with TOP2A deletion

22. Survival P = 0.009 P = 0.002

23. Hazards Ratio Odds ratio for amplification was 2.689 (p=0.039, 95% [CI] of 1.049 to 6.890), whereas for the NPI, it was 2.138 (p=0.000, 95% CI of 1.602 to 2.853) –indicating that TOP2A gene amplification is associated with an increase in the risk of death from the tumour in these cases

24. Conclusion TOP2A is a good independent predictor of survival, equalling or bettering the Nottingham Prognostic Index TOP2A FISH and CISH techniques show excellent agreement Work needs to be carried to find out how many cases in this study were Her-2+

25. Future Work Need for a classification system that can be applied to a heterogeneous population –Ideally, a classification system is required that can be applied to all patients to determine their prognosis and devise treatment strategies tailored according to the patient –will also reduce over-treatment, which is occurring now, because there are unknown factors causing the patient to become unresponsive to a particular treatment Objective for studies like this is to determine a molecular classification so that new drugs can be developed to target over- or under-expressed genes & increase survival chances for patients with breast cancer

26. Any Questions? ?