RB-pathway disruption in breast cancer: differential association with disease subtypes, disease-specific prognosis and therapeutic response

Abstract

In breast cancer, inactivation of the RB tumor suppressor gene is believed to occur via multiple mechanisms to facilitate tumorigenesis. However, the prognostic and predictive value of RB status in disease-specific clinical outcomes has remained uncertain. We investigated RB pathway deregulation in the context of both ER-positive and ER-negative disease using combined microarray datasets encompassing over 900 breast cancer patient samples. Disease-specific characteristics of RB pathway deregulation were investigated in this dataset by evaluating correlation among pathway genes as well as differential expression across patient tumor populations defined by ER status. Survival analysis among these breast cancer samples demonstrates that the RB-loss signature is associated with poor disease outcome within several independent cohorts. Within the ER-negative subpopulation, the RB-loss signature is associated with improved response to chemotherapy and longer relapse-free survival. Additionally, while individual genes in the RB target signature closely reproduce its prognostic value, they also serve to predict and monitor response to therapeutic compounds, such as the cytostatic agent PD-0332991. These results indicate that the RB-loss signature expression is associated with poor outcome in breast cancer, but predicts improved response to chemotherapy based on data in ER-negative populations. While the RB-loss signature, as a whole, demonstrates prognostic and predictive utility, a small subset of markers could be sufficient to stratify patients based on RB function and inform the selection of appropriate therapeutic regimens.

Figure 1

Relative expression of the RB target signature and correlated genes in normal and tumor samples. Correlation between genes in the RB/CCND1 pathway and the RB-loss signature magnitude are shown along side their expression level heat maps in (A) normal breast, (B) all tumor samples and subsets for (C) ER-positive and (D) ER-negative samples. Boxplots show the (E–H) relative transcript expression levels of RB target signature and RB/CCND1 pathway genes. (I) ER-positive and ER-negative human breast cancer cell lysates were subjected to immunoblotting with the indicated antibodies for RB pathway members. (J) AQUA immunohistochemical co-staining of tumor specimens for indicated proteins. Cytokeratin (green) was used to detect epithelial cells. Dapi (blue) staining was utilized to detect all nuclei in the section. Target proteins are stained in red. (K–M) relative protein levels of the RB target signature and RB/CCND1 pathway genes for healthy breast, all tumor samples and tumor samples stratified by ER status.

Figure 2

Correlation between the RB target signature and signatures representing cell proliferation and histological tumor grade. (A) A Venn diagram representing overlap between the RB target signature, the proliferation signature and GGI signature. (B–E) The expression of RB-loss, proliferation and GGI signatures is highly correlated.

Figure 3

Relapse-free survival in subpopulations defined by RB-loss signature expression. Survival curves are shown for low, intermediate and high RB-loss signature magnitude for (A) all tumor samples, (B) the ER-positive subset and (C) the ER-negative subset. A comparison of ER-positive and ERnegative survival curves is shown in (D), using cut offs established in the ER-negative population.

Figure 4

Relapse-free survival in the ER-positive population, stratified by treatment status and RB-loss signature. (A) In ER-positive samples with surgery alone, poor outcome is associated with high RB-loss signature [51% 10 yr relapse-free survival (rfs)] versus low RB-loss signature (95% 10 yr rfs). (B) In ER-positive patients treated with Tamoxifen, outcome in high RB-loss signature samples is improved (66% 10 yr rfs) relative to low RB-loss signature samples (92% 10 yr rfs).

Figure 5

Disease outcomes related to the RB-loss signature and treatment regimens in the ER-negative population. (A) High RB-loss signature expression is associated with different outcomes in ER-positive vs. ER-negative. (B) High RB-loss signature expression is associated with similar survival profiles between ER-positive and untreated ER-negative samples. (C) Chemotherapy is associated with better outcome in ER-negative samples with high RB-loss signature expression. (D) The proportion of pCR and PD in high versus low RB-loss signature groups of ER-positive patients receiving FAC therapy. (E) The proportion of pCR and PD in high versus low RB-loss signature groups of ER-negative patients receiving FAC therapy. (F) The proportion of pCR, pPR and PD in high versus low RB-loss signature groups of ER-negative patients receiving cisplatin therapy.

Figure 6

Candidate markers for targeted therapy. (A) Human breast cancer cell lines were treated with 500 nM PD-0332991 or DMSO control for 24 hours. Percentage BrdU positive (of total) is shown. (B) Human breast cancer cell lines were treated with 500 nM PD-0332991 or DMSO control for 24 hours. Cell lysates were subjected to immunoblot analysis for indicated proteins. (C) Relapse-free survival curves for breast cancer populations stratified by individual gene transcript levels.