Use of fine-needle aspirates for the identification of mutations within the estrogen receptor-alpha hormone-binding domain in tamoxifen-resistant human breast carcinomas

We investigated the hypothesis that mutations within estrogen receptor-alph a (ER alpha) may be one of the mechanisms of breast carcinoma progression t o a hormone-independent phenotype. Fine-needle aspirations were collected f rom patients with tamoxifen resistant metastatic breast carcinomas. Total R NA was isolated and the entire ER alpha hormone-binding domain (HBD) amplif ied by RT-PCR using 4 pairs of overlapping primers. PCR products were scree ned for mutations by single-stranded conformational polymorphism (SSCP) ana lysis and DNA sequencing. Although no mutations were detected by SSCP analy sis, two silent polymorphisms were identified by automated DNA sequencing a t codons 538 [GAC --> GAT (Asp)] and 594 [ACA --> ACG (Thr)]. The codon 538 variant was detected in tumour specimens at a low frequency whereas the co don 594 variant was detected in patients and healthy controls at a similar Frequency. Exon 7-deleted variant was also detected in all tumour specimens . Fine-needle aspirations, in conjunction with RT-PCR, could be a rapid and reliable method for the mutational analysis of the ER alpha HBD. However, mutations within the ER alpha HBD do not appear to represent a significant mechanism in the development of antiestrogen resistance.