Background:The HER-2 receptor undergoes a proteolytic cleavage generating an NH 2 -terminally truncated fragment, p95HER-2, that is membrane-associated and tyrosine-phosphorylated.We have reported that p95HER-2, but not the full-length receptor, p185HER-2, correlated with the extent of lymph node involvement in patients with breast cancer and its expression was significantly enhanced in nodal metastatic tissue. These facts suggested an important role for p95HER-2 either as a marker or cause of metastasis and poor outcome in breast cancer. In this work, we have studied the prognostic value of p95HER-2 in breast cancer. Methods: Primary breast tumor tissues (n = 483) were from surgical resections conducted in hospitals in two different countries: the U.S. (n = 334) and Spain (n = 149). HER-2 protein forms, including p185HER-2 and p95HER-2, were examined in extracts of primary breast tumors by Western blot analysis. The levels of the two forms (high or low) were tested for association with other clinicopathologic factors and for correlation with disease-free survival. Results:The median follow-up was 46 months. A high level of p95HER-2 in primary tumor tissue correlated with reduced 5-year disease-free survival (hazard ratio, 2.55; 95% confidence interval, 2.13-8.01; P < 0.0001). The median time for disease-free survival was 32 versus 139 months in patients with low levels of p95HER-2. In comparison, high levels of the full-length p185HER-2 did not significantly correlate with poor outcome (P > 0.1). Multivariate analysis revealed that high p95HER-2 was an independent predictor of disease-free survival (hazard ratio, 1.59; 95% confidence interval, 1.246-1.990; P = 0.0004). Conclusions: p95HER-2 expression is an independent prognostic factor in breast cancer and defines a group of patients with HER-2-positive breast cancer with significantly worse outcome.

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Although androgens exert major effects on bone remodeling, the mechanisms by which they exert their effects remain unclear. Recently, it has become apparent that receptors for sex steroids may be present in osteoblastic cells. We have examined several cell lines with osteoblastic phenotypes to determine if specific, high affinity androgen receptors are present. Two cell lines of human origin (Saos-2 and U2-OS) and one of rat origin (UMR-106.01) were studied. Androgen binding sites were present in all cell lines. Binding affinities were high (KD = 1.6 - 2.5 x 10(-10) M), and similar to those in classical androgen target tissues (prostate, kidney). Concentrations were greater in the human cell lines (1277 and 1605 sites/cell) than in the rodent line (74 sites/cell). In the human cell lines androgen binding was also specific and typical of androgen receptors in other tissues. Specific estrogen binding was not present in the UMR-106.01 cells, and no estrogen receptors were detectable in the human cell lines using an enzyme-linked receptor immunoassay. Specific binding for progesterone was also absent in the UMR-106.01 cells, but progesterone receptors were detected immunologically in the Saos-2 (119 sites/cell) and U2-OS (118 sites/cell) lines. These findings indicate the presence of androgen receptors that are of similar character to those in classical androgen target tissues, and suggest that the study of these cell lines may be useful in the study of the regulation of androgen effects in osteoblasts.

The growth factor receptor-bound protein-7 gene (GRB7) encodes a multi-domain signal transduction molecule. The purpose of this study was to examine the clinical significance of GRB7 protein expression in human breast cancer. Western blotting analysis of protein extracts from 563 annotated frozen breast tumors was performed. Expression status of GRB7 and HER-2 was correlated with clinical covariates and outcomes. Cox proportional hazards were used to identify factors associated with breast cancer-free interval. The median follow-up was 71 months. P values <0.05 were considered statistically significant (two-sided). A discrepancy between HER-2 and GRB7 protein over-expression was observed. GRB7 protein over-expression was associated with negative estrogen and progesterone receptor status, higher tumor grade, larger primary tumor size, (more) axillary lymph node involvement, higher clinical stage, and shortened breast cancer-free interval. HER-2 protein over-expression was associated only with higher tumor grade. Multi-variate analysis revealed that GRB7 protein over-expression was an independent adverse prognostic factor for breast cancer-free interval (hazard ratio 1.69, 95% confidence interval 1.07-2.67; P = 0.024). The same was true of the subset of patients who did not receive any adjuvant systemic therapy (hazard ratio 1.68, 95% confidence interval 1.16-2.31; P = 0.0055). Using FISH analysis, 32/32 (100%; 95% CI 89-100%) tumors which over-expressed both HER-2 and GRB7 proteins and 1/35 (3%; 95% CI 0-15%) tumors with HER-2 but no GRB7 protein over-expression with Western blotting analysis demonstrated HER-2 gene amplification. GRB7 protein over-expression is an independent adverse prognostic factor in human breast cancer.