A randomised controlled phase II trial of pre-operative celecoxib treatment reveals anti-tumour transcriptional response in primary breast cancer

Abstract

Introduction: Cyclooxygenase-2 (COX-2) is frequently over-expressed in primary breast cancer. In transgenic breast cancer models, over-expression of COX-2 leads to tumour formation while COX-2 inhibition exerts anti-tumour effects in breast cancer cell lines. To further determine the effect of COX-2 inhibition in primary breast cancer, we aimed to identify transcriptional changes in breast cancer tissues of patients treated with the selective COX-2 inhibitor celecoxib.

Methods: In a single-centre double-blind phase II study, thirty-seven breast cancer patients were randomised to receive either pre-operative celecoxib (400 mg) twice daily for two to three weeks (n = 22) or a placebo according to the same schedule (n = 15). Gene expression in fresh-frozen pre-surgical biopsies (before treatment) and surgical excision specimens (after treatment) was profiled by using Affymetrix arrays. Differentially expressed genes and altered pathways were bioinformatically identified. Expression of selected genes was validated by quantitative PCR (qPCR). Immunohistochemical protein expression analyses of the proliferation marker Ki-67, the apoptosis marker cleaved caspase-3 and the neo-angiogenesis marker CD34 served to evaluate biological response.

Results: We identified 972 and 586 significantly up- and down-regulated genes, respectively, in celecoxib-treated specimens. Significant expression changes in six out of eight genes could be validated by qPCR. Pathway analyses revealed over-representation of deregulated genes in the networks of proliferation, cell cycle, extracellular matrix biology, and inflammatory immune response. The Ki-67 mean change relative to baseline was -29.1% (P = 0.019) and -8.2% (P = 0.384) in the treatment and control arm, respectively. Between treatment groups, the change in Ki-67 was statistically significant (P = 0.029). Cleaved caspase-3 and CD34 expression were not significantly different between the celecoxib-treated and placebo-treated groups.

Conclusions: Short-term COX-2 inhibition by celecoxib induces transcriptional programs supporting anti-tumour activity in primary breast cancer tissue. The impact on proliferation-associated genes is reflected by a reduction of Ki-67 positive cells. Therefore, COX-2 inhibition should be considered as a treatment strategy for further clinical testing in primary breast cancer.

Trial registration: ClinicalTrials.gov NCT01695226.

Figures

Figure 1

Flow diagram of the presented study. The design is a double-blind, randomised, controlled phase II trial of pre-operative celecoxib versus placebo in early breast cancer. Note that eight patients had discontinued intervention in the treatment arm. Gene expression profiling (GEP) has been performed from samples where indicated.

Figure 2

qPCR validation of selected genes differentially expressed in celecoxib-treated samples as determined by microarray analysis. Fold-change and the 95% CI (error bars) are shown. Expression of six out of eight genes analysed (indicated by asterisks) was significantly changed in agreement with the microarray analysis.

Figure 3

Effects of celecoxib treatment on cell cycle and proliferation. Contributed map from GenMAPP software with an overview of the pathways and genes involved in cell cycle regulation. The expression fold-changes of each gene are indicated next to the gene box. Genes highlighted in red and green in the left box half represent genes with fold-changes increased and decreased, respectively. Red colour in the right box half indicates a significant change. Grey boxes correspond to genes that were not analysed in the arrays.

Figure 4

Effects of celecoxib treatment on extracellular matrix protein degradation. Map designed on GenMAPP software with an overview of the genes involved in the extracellular matrix protein degradation process. The expression fold-changes of each gene are indicated next to the gene box. Genes highlighted in red and green in the left box half represent genes with fold-changes increased and decreased, respectively. Red colour in the right box half indicates a significant change. Grey boxes correspond to genes that were not analysed in the arrays.

Figure 5

Effects of celecoxib treatment on Ki-67 protein expression. (A) Examples of immunohistochemical staining of nuclear Ki-67 protein on breast cancer tissues yielding a high score (left) and low score (right). Scale bar = 100 μm. (B) Shown are the Ki-67 scores from individual patients in the control arm (plot on left-hand side) and treatment arm (plot on right-hand side). Geometric means in the control group were statistically not different (P = 0.384), while the geometric mean after celecoxib treatment was significantly reduced (P = 0.019). Also, the change of the means between both groups was significantly greater in the celecoxib-treated group (P = 0.029).