Abstract C19: High CCL2 protein expression is associated with poor outcome in ovarian cancer

Abstract

Background: CCL2 is a potent chemoattractant for tumor-associated macrophages, which promotes tumor growth and metastasis. The CCL2/CCR2 interaction plays a key role in promoting tumor inflammation, angiogenesis, proliferation and metastasis. Ovarian tumors and tumor lines secrete CCL2, however, very little is known about the expression of CCR2 and macrophage markers in ovarian tumors. Using a Yale University ovarian cancer tissue microarray cohort with associated outcome data, we analyzed the expression of CCL2, CCR2, and macrophage markers CD68, iNOS (M1 marker) and Arginase-1 (M2 marker) using fluorescence immunohistochemistry methods. Methods: The Yale YTMA18-2 Ovarian Cancer tissue microarray cohort consists of 239 stage I–IV ovarian tumors of all major histological subtypes and also included 12 normal gynecologic tissue samples for comparison purposes. The expression levels were assessed using Automated QUantitative Analysis (AQUA® technology, HistoRx Inc.) and were correlated with clinical outcome. CCR2 and CCL2 were measured within pan-cytokeratin defined cytoplasmic regions, and CD68, iNOS and Arginase-1 were measured within the entire sample regions. These biomarkers were analyzed for relationships with each other by Spearman Rho analysis and unsupervised hierarchical clustering, and for association with outcome by Cox univariate analysis and Kaplan-Meier analysis. Punch cores from the tumor sample utilized in the ovarian cancer tissue microarray cohort were also analyzed for genotyping five SNPs in the CCL2 promoter region. Results: There was no significant difference in CCL2, CCR2, CD68, iNOS (M1 marker) and Arginase-1 (M2 marker) expression by age group, stage or major histology grouping (all p>0.05). When looking at all tumor tissues, there was a significant difference in patient outcome (10 year and full time period, p = 0.028 and p = 0.047, respectively) based on separation by CCL2 cluster-based expression groupings. High CCL2 expression patients have a worse outcome, with a 1.9 fold higher risk of death at 5 years than other patients in categorical Cox univariate analysis. Although there was no correlation between CCR2 and CCL2, Kaplan-Meier analysis of unsupervised cluster groupings based on both markers identified a subset of ovarian cancer patients with high expression of CCL2 and intermediate/high expression of CCR2 that have the poorest survival compared with other groupings. Additionally, ovarian tumor tissues expressed 3.5-fold higher level of CCL2 and 1.2 -fold higher levels of CCR2 (mean AQUA scores, respectively) compared to normal samples (p< .0001). There were no significant differences in patient outcome by CD68, iNOS (M1 marker) and Arginase-1 (M2 marker)cluster groupings. There was a statistically significant difference in patient outcome by Kaplan-Meier analysis between two SNPs but no correlation with CCL2 expression was observed. Conclusion: Taken together, these data suggest that there is a subset of ovarian cancer patients with elevated CCL2 protein expression levels associated with poor outcome. This is the first study to show a link between high CCL2 protein expression levels and poor survival in ovarian cancer. Thus, additional exploration of CCL2 expression levels as a predictive biomarker in ovarian cancer is warranted. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C19.