TOP2A IS AN INDEPENDENT PREDICTOR OF SURVIVAL IN UNSELECTED BREAST CANCER

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

• Amit Pancholi

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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 adenocarcinomas

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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 lt 3.4
• Moderate MPG NPI 3.4 5.4
• Poor PPG NPI gt 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
  (survivalgt5yrs)
• Luminal B. ER with poor prognosis
  (survivallt5yrs)
• 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 IIa 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
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• 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 redgreen signals determined aberration
  status
• E.g. gt2 is amplification, lt0.8 is deletion

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CISH

• Colorimetric in situ hybridisation
• Digoxigenin-labelled probe
• gt5 signals per cell in gt50 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
• 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

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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 (plt0.05), distant
  metastases (plt0.03) and tumour recurrence
  (plt0.015)
• No association with TOP2A deletion

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Survival

P 0.002
P 0.009
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Hazards Ratio

• Odds ratio for amplification was 2.689 (p0.039,
  95 CI of 1.049 to 6.890), whereas for the NPI,
  it was 2.138 (p0.000, 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?

• ?