VEGFA Amplification/Deletion in Human Breast Tumors
Bryan P Schneider, Milan Radovich, Bradley Hancock, Nawal Kassem, George Sledge, Kirsten Vang Nielsen, Sven Muller, Mangesh Thorat, Rutika Mehta, Sunil Badve. Indiana University School of Medicine, Indianapolis, IN; Dako A/S, Glostrup, Denmark
Background: The anti-VEGF antibody, bevacizumab, has been FDA approved for the treatment of breast cancer. While germline variability (ie, single nucleotide polymorphisms) may serve as a predictive marker for anti-VEGF therapy, to date tumor-specific variability has not. More specifically, amplification or deletion of the VEGFA gene has not been studied or evaluated as a predictive or prognostic biomarker for breast cancer.
Design: A VEGFA/centromere enumerization-6 (CEN-6) probe was created and validated using DNA clones and restriction enzyme fragment measurements. The final product contained a RP11-710-L16 probe covering 183 KB including the VEGFA gene and flanking regions. The CEN-6 probe was developed with fluorescein isothiocyanate labeled peptide nucleic acid oligonucleotides. VEGFA and CEN-6 probes were tested on metaphase spreads to exclude cross-hybridization to other chromosomes.
A tissue microarray containing 93 breast cancers was analyzed for the presence of VEGFA gene amplification and/or deletion using a FISH protocol similar to the TOP2A FISH pharmDx™ Kit. VEGFA/CEN-6 signal ratio in cancer cells was recorded. Normal cells in the tissue sections served as an internal positive control of pretreatment and hybridization efficiency. A ratio of <0.8 was considered deleted, ≥1.5–1.99 was considered borderline amplified, whereas a ratio of ≥2.0 was considered amplified.
Results: Of the 93 core tissue specimens, FISH analysis was successful in 80 cases (86%) of the cases. Of these, 11% were found to demonstrate VEGFA deletions, 9% were borderline amplified, and 5% were amplified. Thus, 25% in total had genetic aberrations of this gene. The aberrations did not correlate with ER or HER2 expression.