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Single-nucleotide polymorphisms (SNPs) can be assayed using DNA isolated from archival formalin-fixed, paraffin-embedded (FFPE) samples, making retrospective pharmacogenetic studies possible. In this study, we describe methods that significantly increase the number of SNP determinations possible using FFPE samples. Quantifying the amount of DNA amenable to PCR (amplification-quality DNA, AQ-DNA) allows a significant reduction in the amount of sample required for Taqman-based SNP assays. Optimizing AQ-DNA input increases PCR amplification efficiency and SNP determination accuracy. DNA was extracted from 39 FFPE tumor sections and matched tumor and stromal cores, which were of the type used to generate tissue microarrays. Sections and tumor cores yielded sufficient AQ-DNA for more than 1000 SNP determinations. Seven SNPs were assessed following individual assay optimization for minimal AQ-DNA. Genotypes from tumor cores for single SNPs were 92.3-100% concordant with those obtained from sections. Using these methods, the number of SNP genotypes that can be determined from single FFPE samples is greatly increased expanding the genetic association studies possible from limited archival specimens. The use of tumor cores is of particular importance as the harvesting of tumor cores has minimal impact on the utility of the donor blocks for other purposes. [ABSTRACT FROM AUTHOR]

Objectives Efavirenz-based HIV therapy is associated with breast hypertrophy and gynaecomastia. Here, we tested the hypothesis that efavirenz induces gynaecomastia through direct binding and modulation of the oestrogen receptor (ER). Methods To determine the effect of efavirenz on growth, the oestrogen-dependent, ER-positive breast cancer cell lines MCF-7, T47D and ZR-75-1 were treated with efavirenz under oestrogen-free conditions in the presence or absence of the anti-oestrogen ICI 182,780. Cells treated with 17β-oestradiol in the absence or presence of ICI 182,780 served as positive and negative controls, respectively. Cellular growth was assayed using the crystal violet staining method and an in vitro receptor binding assay was used to measure the ER binding affinity of efavirenz. Results Efavirenz induced growth in MCF-7 cells with an estimated effective concentration for half-maximal growth (EC50) of 15.7 μM. This growth was reversed by ICI 182,780. Further, efavirenz binds directly to the ER [inhibitory concentration for half maximal binding (IC50) of ∼52 μM] at a roughly 1000-fold higher concentration than observed with 17β-oestradiol. Conclusions Our data suggest that efavirenz-induced gynaecomastia may be caused, at least in part, by drug-induced ER activation in breast tissues. [ABSTRACT FROM AUTHOR]

The selective estrogen receptor modulator tamoxifen is routinely used for treatment and prevention of estrogen-receptor-positive breast cancer. Studies of tamoxifen adherence suggest that over half of patients discontinue treatment before the recommended 5 years. We hypothesized that polymorphisms in CYP2D6, the enzyme responsible for tamoxifen activation, predict for tamoxifen discontinuation. Tamoxifen-treated women (n=297) were genotyped for CYP2D6 variants and assigned a ‘score’ based on predicted allele activities from 0 (no activity) to 2 (high activity). Correlation between CYP2D6 score and discontinuation rates at 4 months was tested. We observed a strong nonlinear correlation between higher CYP2D6 score and increased rates of discontinuation (r2=0.935, P=0.018). These data suggest that presence of active CYP2D6 alleles may predict for higher likelihood of tamoxifen discontinuation. Therefore, patients who may be most likely to benefit from tamoxifen may paradoxically be most likely to discontinue treatment prematurely.The Pharmacogenomics Journal (2009) 9, 258–264; doi:10.1038/tpj.2009.14; published online 5 May 2009 [ABSTRACT FROM AUTHOR]

Invasive lobular carcinoma (ILC) represents ^10% of newly diagnosed breast tumors, accounting for -30,000 cases annually in the US. However, ILC has been understudied as a breast cancer subtype. ILC-specific signaling pathways and responses to endocrine therapies are not well characterized. Recent retrospective analyses of luminal-type breast cancers suggest that ILC patients treated with endocrine therapy have poorer disease-free survival and overall survival than invasive ductal carcinoma (IDC) patients with similar biomarkers. We hypothesize that unique transcriptional control of estrogen receptor-alpha (ERa) by estrogens and anti-estrogens in ILC cells drive a differential response of ILC tumors to endocrine therapies. The ILC cell lines MDA MB 134VI and SUM44PE were used as in vitro models of ILC tumors to examine responses to estradiol (E2) and the anti-estrogens tamoxifen (Tarn), 4-hydroxytamoxifen (4OHT), endoxifen (Bx), and fulvestrant (ICI). We examined cell growth and expression of canonical ERa-regulated genes in response to E2. Cells were also treated with anti-estrogens +/- E2 to examine their ability to inhibit E2-induced growth. To establish a genome-wide profile of ERa-mediated gene regulation in ILC cells, ILC cells were subjected to gene expression microarray analysis following 0-24hrs treatment with 1 nM E2. In parallel to these in vitro studies, in vivo models of ILC are being assessed for endocrine responsiveness, including MDA MB 134VI xenografts and the primary human tumor xenograft HCI-013. We observed that both MDA MB 134VI and SUM44PE are growth-induced by E2 treatment. However, both cell lines also present de novo resistance to Tarn, 4OHT, and Bx. While ICI treatment completely blocked E2-induced growth in MDA MB 134VI, Tarn, 4OHT, and Bx acted as partial agonists. Treatment with these compounds alone induced "-25% growth relative to E2, and E2-induced growth could only be inhibited -75%. Similarly, SUM44PE cells are strongly growth-inhibited by ICI treatment, whereas growth is not reduced by Tarn, 4OHT, or Bx treatment. Consistent with these observations, novel patterns of gene expression are induced by E2 treatment in ILC cells. E2-t.reat.ment induced canonical targets (e.g. GREB1, IGFBP4) in both ILC cell lines. However, in MDA MB 134VI cells only -35% of genes regulated by E2 at 24hrs overlap with those regulated in the IDC cell line MCF-7. Further, a subset of the overlapping genes is differentially regulated between cell types, including ESR1 (1.25-fold and 0.67-fold versus control in MDA MB 134VI and MCF-7, respectively), HOXC6, PMP22, and L1CAM. Examination of these observations in in vivo models is currently ongoing. These data are consistent with the hypothesis that E2 and anti-estrogens differentially regulate ERa-mediated gene expression in ILC cells versus IDC cells. The de novo resistance to tamoxifen observed in ILC cells may correlate with the worse outcomes in ILC patients observed in retrospective analyses. We hypothesize that elucidation of the mechanisms responsible for differential ERa regulation may reveal novel therapeutic targets for treating ILC patients and overcoming endocrine resistance. [ABSTRACT FROM AUTHOR]