The antiestrogen tamoxifen, a major endocrine therapy of estrogen receptor (ER)-positive breast cancer, is nevertheless inefficient in 30 to 40% of cases for unknown reasons. We retrospectively studied 50 ER-positive primary breast carcinomas. All of the patients had received tamoxifen as the only adjuvant therapy. They were divided into two groups depending on whether they relapsed within 5 years (16 tamoxifen-resistant cases) or did not relapse within 5 years (34 tamoxifen-sensitive cases). The expression of total ER␤ protein, and of ER␤cx protein, was estimated anonymously in formalin-fixed, paraffin-embedded tumor sections, by using specific antibodies and quantifiying nuclear immunostaining with a computer image analyzer. All of the tumors were found to be HER-2/neu-negative by immunohistochemistry.Univariate analysis showed that Scarff-Bloom-Richardsson grade modified by Elston (SBR grade; P < 0.001), tumor size (P ‫؍‬ 0.042), and MIB-1 proliferation index (P ‫؍‬ 0.02) were significantly higher in tamoxifen-resistant tumors. A low level of total ER␤, whether in percentage of positive cells or in quantitative immunocytochemical (QIC) score, was also associated with tamoxifen resistance (P ‫؍‬ 0.004). ER␤cx expression and lymph node status were similar between the two groups. The expression of ER␤ in the total population was positively correlated with ER␤cx (r ‫؍‬ 0.63, P < 0.001), and was independent of the other parameters. In a multivariate analysis, ER␤ expression was the most important variable (P ‫؍‬ 0.001), followed by SBR grade (I؉II versus III; P ‫؍‬ 0.008), and MIB-1 (P ‫؍‬ 0.016).To conclude, tamoxifen resistance is associated with classical variables of aggressive tumors (high SBR grade, proliferation index, and tumor size) but not with node invasiveness. Low ER␤ level is an additional independent marker, better than ER␣ level, to predict tamoxifen resistance.

Identification of proteins that markedly vary during early steps of mammary carcinogenesis may help to understand its pathophysiology and to develop a prevention strategy. The expression of total estrogen receptor h (ERh) protein and of its COOH-terminally spliced variant ERhcx (or ERh2) was compared in 43 invasive breast cancers and in 39 adjacent normal mammary glands and 26 ductal carcinoma in situ (DCIS). Thirty-six breast cancers were ER positive by radioligand binding assay. The analysis was done by immunohistochemistry on adjacent sections of formalin-fixed, paraffin-embedded tumors using polyclonal anti-ERh 503 IgYand sheep polyclonal ERhcx antibodies that were previously validated. Nuclear staining was quantified using a computerized image analyzer in selected areas of normal and cancer epithelial cells.Total ERh expression was high in normal glands, decreased in DCIS (P = 0.0004), and increased from DCIS to invasive tumors (P = 0.029). In contrast, the ERhcx expression was low in normal glands, increased significantly in DCIS (P = 0.0014), and continued to increase in invasive carcinomas (P = 0.0027) in both ERa-positive and ERa-negative tumors. This is the first study showing a significant increase of the ERhcx variant protein in DCIS and invasive breast cancer compared with adjacent normal glands.This contrasts with the decrease of the total ERh level in the same patients and indicates different mechanisms to explain these variations during mammary carcinogenesis. It also suggests a role of the ERhcx variant in carcinogenesis opposite to the protective effect of the wild-type ERh1.

Progestins increase the risk of breast cancer in the hormone therapy of menopause, and progesterone receptor-induced fatty acid synthase (FAS) is a potential therapeutical target of breast cancer. In a first attempt to specify in which lesions at risk of breast cancer progestins might be acting, we have compared the progesterone receptor (PR) and FAS expression in preinvasive breast lesions and in adjacent ''normal'' mammary glands. We used archive paraffin-embedded tissues from 116 patients, with 164 lesions of increasing histological risk from nonproliferative ''benign'' breast disease (BBD) to in situ breast carcinomas. Immunostaining using our FAS antibody and a PR antibody from Dako was quantified as continuous variables by computer-assisted image analysis. FAS level increased (p < 10 23 by the KruskallWallis test) in all lesions, starting from nonproliferative BBD, and was maximal in in situ carcinoma. The % of PR-positive cells increased from nonproliferative BBD and was higher in proliferative atypia (p < 10 23 ). It was very low in high-grade DCIS corresponding to a likely different carcinogenesis pathway. There was a trend for a positive correlation between FAS and PR in normal glands. However, the 2 markers increased independently in BBD and were negatively correlated in in situ carcinomas. FAS and PR were positively correlated with Ki67 in BBD. The increased PR level in premalignant steps of mammary carcinogenesis suggests an early increased responsiveness to progestins. The increased FAS expression, in lesions parallel to their increased breast cancer risk, suggests further studies to develop new markers of high-risk lesions and to prevent breast cancer. ' 2006 Wiley-Liss, Inc.

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