Quantitative progesterone receptor expression and efficacy of anti-estrogen therapy in breast cancer.

The central role of estrogen receptor (ER) presence in predicting which breast cancer patients are likely to benefit from anti-estrogen therapies is well-established, but the added benefit of progesterone receptor (PR) and in particular low levels of PR is less well understood. The objective of this study was to determine the quantitative relationship between borderline levels of PR and subsequent benefit from anti-estrogen therapy. We examined data from 447 patients, age 50 or older. ER and PR levels were quantitated by conventional ligand binding assay and Scatchard plot analysis or by enzyme-linked immunoassay. Comparison of clinical outcome in relation with ER and PR status was calculated using Kaplan-Meier actuarial survival analysis and the log-rank test. Subpopulation treatment effect pattern plot (STEPP) analysis was used to explore the interaction between treatment effects and ER or PR levels for the 409 patients with ER values greater than 0. For anti-estrogen treated patients, when the ER and PR positivity cut-off was set at 1.0 fmole/mg protein, there was a statistically significant advantage for patients with ER+PR+ over ER+ PR- tumors for both breast cancer-free interval (BCFI) and overall survival (OS). STEPP analysis found no overall interaction between treatment outcome (5 year survival probability) and levels of hormone receptor. However, patients with borderline PR levels did not appear to benefit from anti-estrogen therapy. PR levels above borderline in addition to the presence of ER predicts an increased probability of benefit from anti-estrogen therapy in breast cancer patients.

GRB7 protein over-expression and clinical outcome in breast cancer.

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.

HER-2 gene amplification in human breast cancer without concurrent HER-2 over-expression.

BACKGROUND: Testing for human epidermal growth factor receptor-2 (HER-2) in breast cancer is performed by either immunohistochemistry (IHC) or in situ hybridization (ISH). The growth factor receptor-bound protein-7 (GRB7) gene is in close proximity to HER-2 on chromosome 17q11-12 and codes a signal transduction molecule shown to be an independent adverse marker in breast cancer. METHODS: HER-2 and GRB7 protein expression from 613 frozen breast tumors was determined by Western analysis. HER-2 protein results were confirmed with IHC. Commercial HER-2 FISH was performed on a subset of tumors with multi-probe FISH used to assess the extent of HER-2 gene amplification. mRNA expression was determined by Multi-plex RT-PCR. RESULTS: Seven tumors with GRB7 protein over-expression scored HER-2 FISH amplified but had no HER-2 protein over-expression. Four of the 7 tumors showed elevated GRB7 but not HER-2 mRNA over-expression. The breast cancer cell line HCC3153 did not over-express HER-2 protein but showed HER-2 FISH amplification of a limited segment around the HER-2 gene. Ten breast cancer tumors from the TCGA database had gene copy number increases around HER-2 without HER-2 mRNA or protein over-expression. CONCLUSIONS: A subset of human breast cancers that test positive with FISH for HER-2 gene amplification do not over-express HER-2 protein. One mechanism for this discordance is the incomplete amplification of the smallest HER-2 region of chromosome 17q11-12, which includes GRB7. HER-2 gene amplification without protein over-expression is clinically significant because patients with such tumors are unlikely to benefit from HER-2 targeted therapy.