Polymorphism Interaction to Predict Bevacizumab Efficacy
Condition: Metastatic Breast Cancer
MeSH terms: Breast Neoplasms
Study type: Observational

Summary: 
      Although many attempts have been done to identify vascular endothelial growth factor-A
      (VEGF-A) single nucleotide polymorphisms (SNPs) correlated with bevacizumab response, in
      advanced cancer patients, the results are still inconclusive.

      We will conduct a pharmacogenetic study to assess, in a population of metastatic breast
      cancer (MBC) patients, the possible predictive role of VEGF-A, VEGF receptor-2 (VEGFR-2),
      interleukin-8 (IL-8), hypoxia inducible factor-1α (HIF-1α), hypoxia inducible factor-2α
      (HIF-2α) and thrombospondin-1 (TSP-1) SNPs for bevacizumab response when combined with
      first-line paclitaxel and for progression free survival (PFS). Analyses will be performed on
      germline DNA obtained from blood samples and SNPs will be investigated by real-time
      polymerase chain reaction (PCR) technique. The multifactor dimensionality reduction (MDR)
      methodology will be applied to investigate the interaction between SNPs.
    

Detailed description: 
      Metastatic breast cancer (MBC) patients from eight Italian divisions of Medical Oncology,
      with histologically confirmed HER2-negative MBC, treated with a first-line therapy including
      bevacizumab 10 mg/m2 i.v. on days 1 and 15 combined with first-line paclitaxel 90 mg/m2 i.v.
      on days 1, 8 and 15, every 4 weeks, will be enrolled for the present pharmacogenetic study.
      MBC patients treated with a first-line chemotherapy including paclitaxel without bevacizumab
      will be also enrolled as control group.

      Sites of metastatic disease will be radiologically re-evaluated according to the Response
      Evaluation Criteria in Solid Tumors (RECIST) criteria 1.1, in patients with measurable
      disease. In patients without measurable lesions, progression of disease will be defined when
      new lesions appeared or when existing lesions evolved. Likewise, in the case of non
      measurable lesions, deterioration of clinical condition not due to treatment toxicity, will
      be defined as progression of disease.

      Progression-free survival (PFS) will be defined as the period of time from the beginning of
      the treatment to the first observation of disease progression as above described, or death
      from any cause. All patients will be assessed for response, PFS and overall survival. Each
      patient entering the study will sign the informed consent. The protocol has been approved by
      ethic committee of Azienda Ospedaliera-Universitaria Pisana, Pisa, Italy, (CESM 3077/2010).

      Genotyping analyses Blood samples (3 ml) will be collected in ethylenediaminetetraacetic acid
      (EDTA) tubes and stored at -80°C. Genes and polymorphisms involved in the angiogenesis
      pathway and already suggested as predictors of bevacizumab response, will be chosen for the
      present analyses. Germline DNA extraction will be performed using QIAamp DNA Blood Mini Kit
      (Qiagen, Valencia, California, USA). Allelic discrimination of genes will be performed using
      an ABI PRISM 7900 SDS instrument (Applied Biosystems, Carlsbad, California, USA) and with
      validated TaqMan® SNP genotyping assays (Applied Biosystems). PCR reactions will be carried
      out according to the manufacturer's protocol. Genotyping will be not performed until an
      adequate number of events (>80% on study population) will be reported in terms of PFS.

      Statistical analysis The first aim of this retrospective analysis will be to evaluate the
      possible role of these investigated gene polymorphisms to predict the bevacizumab response in
      terms of PFS. The secondary end-points will be the correlations with overall survival (OS)
      and response rate. All polymorphisms will be analyzed for deviation from the Hardy-Weinberg
      Equilibrium (HWE) by means of comparison between observed allelic distributions with those
      expected from the HWE by on χ2 test. Any correlation between gene polymorphisms and response
      rate will be analyzed by the two-sided Fisher's Exact Test. The association between each
      individual polymorphism and the most relevant clinical-pathological characteristics with PFS
      will be tested using a Cox proportional hazards model. The Multifactor Dimensionality
      Reduction (MDR) methodology will be applied (using version 2.0 beta 6 of MDR software
      available on http://sourceforge.net/projects/mdr/) to investigate the role of an interaction
      between gene polymorphisms in identifying biomarkers of paclitaxel plus bevacizumab response.

      The genotype combination with the highest PFS benefit correlated with an OS improvement will
      be chosen for further analyses. The difference in PFS between favourable genetic profiles and
      the unfavourable genetic profiles will be assessed with the log-rank test and the
      Kaplan-Meier method to evaluate survival curves. A Cox proportional hazards model, with the
      possible genetic profiles and the clinical and pathological patient characteristics
      individually correlated with the PFS, will be used to calculate the adjusted hazards ratio
      (HR) and the 95% confidence interval (95% CI). A P value of <0.05 will be accepted as
      statistically significant. The Kaplan-Meier and Cox proportional hazards analyses will be
      performed using the SPSS version 17.0 (SPSS, Chicago, IL).
    

Observational model: Cohort
Time perspective: Retrospective
Sampling method: Non-Probability Sample
Study population: 
        metastatic breast cancer patients from eight Italian divisions of Medical Oncology
      
Biospecimen description: 
      whole blood
    
Keywords: metastatic breast cancer; bevacizumab; single nucleotide polymorphism; VEGF-A; VEGFR-2; IL-8