Interactions between immunity, proliferation and molecular subtype in breast cancer prognosis

Nagalla et al. Genome Biology Interactions between immunity, proliferation and molecular subtype in breast cancer prognosis Srikanth Nagalla Jeff W Chou Mark C Willingham Jimmy Ruiz James P Vaughn 0 Purnima Dubey Timothy L Lash Stephen J Hamilton-Dutoit Jonas Bergh Christos Sotiriou Michael A Black 1 Lance D Miller 0 0 Department of Cancer Biology, Wake Forest School of Medicine, Medical Center Boulevard , Winston-Salem, NC, 27157 , USA 1 Department of Biochemistry, Otago School of Medical Sciences, University of Otago , 710 Cumberland Street, Dunedin, 9016 , New Zealand Background: Gene expression signatures indicative of tumor proliferative capacity and tumor-immune cell interactions have emerged as principal biology-driven predictors of breast cancer outcomes. How these signatures relate to one another in biological and prognostic contexts remains to be clarified. Results: To investigate the relationship between proliferation and immune gene signatures, we analyzed an integrated dataset of 1,954 clinically annotated breast tumor expression profiles randomized into training and test sets to allow two-way discovery and validation of gene-survival associations. Hierarchical clustering revealed a large cluster of distant metastasis-free survival-associated genes with known immunological functions that further partitioned into three distinct immune metagenes likely reflecting B cells and/or plasma cells; T cells and natural killer cells; and monocytes and/or dendritic cells. A proliferation metagene allowed stratification of cases into proliferation tertiles. The prognostic strength of these metagenes was largely restricted to tumors within the highest proliferation tertile, though intrinsic subtype-specific differences were observed in the intermediate and low proliferation tertiles. In highly proliferative tumors, high tertile immune metagene expression equated with markedly reduced risk of metastasis whereas tumors with low tertile expression of any one of the three immune metagenes were associated with poor outcome despite higher expression of the other two metagenes. Conclusions: These findings suggest that a productive interplay among multiple immune cell types at the tumor site promotes long-term anti-metastatic immunity in a proliferation-dependent manner. The emergence of a subset of effective immune responders among highly proliferative tumors has novel prognostic ramifications. Breast cancer; gene signatures; hierarchical clustering; immune metagene; intrinsic subtypes; metagene tertiles; multivariable analysis; prognosis; proliferation metagene; survival analysis - Background Expression profiling studies in human tumors have enabled new insights into the genes and pathways that contribute to tumorigenesis and spurred the development of gene expression signatures prognostic of patient outcomes. Genes comprising prognostic signatures often provide clues to the pathobiological mechanisms that drive cancer progression. With the aim of discovering genes with statistical associations with breast cancer recurrence, we and others have identified a number of genes with roles in cellular proliferation [1-6], including multi-gene proliferation signatures that directly reflect tumor proliferative capacity [1,4-7]. These signatures are highly significantly associated with poor patient outcomes, consistent with the view that uncontrolled cell proliferation is a central feature of neoplastic disease and, ultimately, a contributing factor in metastatic progression [8,9]. Indeed, proliferation-associated genes are common components of many previously reported prognostic gene signatures, including Genomic Healths 21gene Oncotype Dx test [10,11] (Genomic Health, Inc., Redwood City, CA, USA), and frequently account for the majority of the prognostic power driving the performance of these signatures [12-14]. Thus, a clear biological understanding of how prognostic genes relate to different aspects of tumor pathobiology is imperative to both the optimal construction of prognostic models and the elucidation of key regulators of cancer behavior. In recent years, we and others have observed that elevated expression levels of many genes involved in immune response pathways are associated with reduced risk of breast cancer recurrence [15-19]. These observations support the view that cancer-leukocyte interactions in the microenvironment of established tumors may function to limit the growth and metastatic progression of breast cancer [20-22]. However, the extent to which these genes reflect different effector cell populations, or contribute to patient prognosis in the presence of other predictive biomarkers such as proliferation, remains unclear. In this report, we investigate the biological origins of coordinately expressed genes in breast cancer that exhibit statistical associations with patient distant metastasis-free survival (DMFS). We identify gene clusters indicative of tumor-immune cell interactions that organize into three distinct immunity-related gene signatures, or metagenes, and shed light on their prognostic implications for tumors of differing proliferative capacity with an emphasis on highly proliferative breast cancers and the most aggressive intrinsic molecular subtypes in particular. Results Reproducible clustering of prognostic genes with immune cell functions To characterize prognostic gene modules, we created a multi-study microarray database of 2,116 breast tumor expression profiles of which 1,954 were annotated with corresponding clinicopathological data including DMFS (See Additional file 1 for clinical details). To facilitate gene discovery, we randomized the dataset across study groups and clinical features into two equivalent patient subpopulations, termed patient groups 977A and 977B (Table 1). In each patient group, Cox proportional hazards regression was conducted to identify genes with statistically significant associations with DMFS while controlling for false discoveries (q < 0.1). The analysis identified 3,094 significant gene probe sets in 977A and 3,304 in 977B (gene details provided in Additional file 2). In parallel, the DMFS-associated genes identified in each patient group were hierarchically clustered to enable analysis of gene correlation structure (Figure 1 and Additional file 3). As anticipated, a proliferation gene cluster was readily identifiable in both patient groups. This cluster of genes has been previously described in multiple studies as being significantly associated with patient survival [1,2,5,23], and consists of the highly correlated group of cell cycle genes associated ER+: Estrogen receptor-positive breast cancer; HER2-E: human epidermal growth factor receptor 2-enriched. with markers of tumor cell proliferation [6,7,24]. In a subset analysis, we examined the correlation between this proliferation gene cluster and clinical markers of proliferation. As expected, we observed a strong positive correlation between the ave (...truncated)