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Estrogen receptor (ER) α is a major determinant of breast tumor progression, and a key target for hormonal-based therapies. Alterations in signal transduction to the ERα pathway have recently been identified as contributing to the failure of hormonal therapy and disease recurrence, however, mutations in the ERα gene have been infrequently identified. We previously reported finding a somatic ERα mutation (A908G) in premalignant breast lesions. Recently, dye-labeled terminator genomic sequencing was also used to screen for the A908G ERα mutation in microdissected human breast specimens from women in the United States, but the mutation was not detected using this methodology (Tebbit et al. 2004). In the current study, we have compared three genomic sequencing approaches (dye-labeled terminator automated fluorescent sequencing, manual Sanger dideoxysequencing, and primer extension minisequencing) with DNA isolated from microdissected invasive breast tumors. We demonstrate that the A908G ERα mutation is indeed present in invasive ductal and lobular breast tumors, and that the primer extension minisequencing approach may be more suitable for the detection of the mutation at this nucleotide. We demonstrate that sequence-dependent incorporation may be complicating the discrimination of the mutant base in both the forward and reverse strand due to uneven base peak heights. Our results suggest that the identification of heterozygotes at this site is possible with the use of a sensitive primer extension minisquencing approach. Using this optimized method, the overall incidence of the mutation was 40% in untreated, primary breast tumors. The presence of the mutation was significantly associated with larger tumor size and positive lymph node status. Correlations between the presence of the A908G ERα mutation, disease-free and overall survival will be presented.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]