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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
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Outline
• Breast Cancer Background
• Classification Systems
• TOP2A Background
• Method
• Results
• Conclusion
• Future Work
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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
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Breast Cancer Morphology
• Mostly adenocarcinomasBreast Cancer
Invasive In Situ
Ductal – 80%
Lobular – 10%
Tubular – 1%
Ductal – 5% Lobular – 6%
Mucoid – 1%
Medullary – 1%
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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
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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
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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
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Molecular Factors
• Oncogene abnormalities– E.g. p53
• HER-2 (erb-B2/neu) oncogene expression– Poorer prognosis
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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.
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Current Classification
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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
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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
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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
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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
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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
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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
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CISH
• Colorimetric in situ hybridisation– Digoxigenin-labelled probe– >5 signals per cell in >50% of cancer cells is
amplification
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Tissue Microarrays
• Revolutionised cancer research
• Allows many patients to be screened in relatively short time
• We had 150 patient-tissue sections (cores) per slide
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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
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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
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Survival
•
Disease Free Interval200150100500
Cu
mu
lati
ve R
ecu
rren
ce
1.0
0.8
0.6
0.4
0.2
0.0
Yes-censoredNo-censoredYesNo
TOPO IIα Gene Amplification
Kaplan-Meier Survival Functions
Overall Survival200150100500
Cu
mu
lati
ve S
urv
ival
1.0
0.8
0.6
0.4
0.2
0.0
Yes-censoredNo-censoredYesNo
TOPO IIα Gene Amplification
Kaplan-Meier Survival Functions
P = 0.009P = 0.002
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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
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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+
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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
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Any Questions?
?