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

Outline

• Breast Cancer Background

• Classification Systems

• TOP2A Background

• Method

• Results

• Conclusion

• Future Work

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

Breast Cancer Morphology

• Mostly adenocarcinomasBreast Cancer

Invasive In Situ

Ductal – 80%

Lobular – 10%

Tubular – 1%

Ductal – 5% Lobular – 6%

Mucoid – 1%

Medullary – 1%

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

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

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

Molecular Factors

• Oncogene abnormalities– E.g. p53

• HER-2 (erb-B2/neu) oncogene expression– Poorer prognosis

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.

Current Classification

Current Classification

• 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

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

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

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

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

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

CISH

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

amplification

Tissue Microarrays

• Revolutionised cancer research

• Allows many patients to be screened in relatively short time

• We had 150 patient-tissue sections (cores) per slide

Results

• Results were analysed in a number of ways– Pearson’s 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

Results

• CISH & FISH correlated significantly (Spearman’s 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

Survival

•

Disease Free Interval200150100500

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TOPO IIα Gene Amplification

Kaplan-Meier Survival Functions

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TOPO IIα Gene Amplification

Kaplan-Meier Survival Functions

P = 0.009P = 0.002

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

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+

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

Any Questions?

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