A signature of immune function genes associated with recurrence-free survival in breast cancer patients

Abstract

The clinical significance of tumor-infiltrating immune cells has been reported in a variety of human carcinomas including breast cancer. However, molecular signature of tumor-infiltrating immune cells and their prognostic value in breast cancer patients remain elusive. We hypothesized that a distinct network of immune function genes at the tumor site can predict a low risk versus high risk of distant relapse in breast cancer patients regardless of the status of ER, PR, or HER-2/neu in their tumors. We conducted retrospective studies in a diverse cohort of breast cancer patients with a 1-5 year tumor relapse versus those with up to 7 years relapse-free survival. The RNAs were extracted from the frozen tumor specimens at the time of diagnosis and subjected to microarray analysis and real-time RT-PCR. Paraffin-embedded tissues were also subjected to immunohistochemistry staining. We determined that a network of immune function genes involved in B cell development, interferon signaling associated with allograft rejection and autoimmune reaction, antigen presentation pathway, and cross talk between adaptive and innate immune responses were exclusively upregulated in patients with relapse-free survival. Among the 299 genes, five genes which included B cell response genes were found to predict with >85% accuracy relapse-free survival. Real-time RT-PCR confirmed the 5-gene prognostic signature that was distinct from an FDA-cleared 70-gene signature of MammaPrint panel and from the Oncotype DX recurrence score assay panel. These data suggest that neoadjuvant immunotherapy in patients with high risk of relapse may reduce tumor recurrence by inducing the immune function genes.

Fig. 1

Unsupervised gene clustering. a Unsupervised cluster visualization of genes differentially expressed among relapse (n = 8) and relapse-free (n = 9) patients. Tumors were hybridized to 36K oligo human array. Genes with at least 80% presence among all samples (9797) were projected using log2 intensity. Red indicates over-expression; green indicates under-expression; black indicates unchanged expression; gray indicates no detection of expression (intensity of both Cy3 and Cy5 below the cutoff value). Each row represents a single gene; each column represents a single sample. The dendrogram at the left of matrix indicates the degree of similarity among the genes examined by expression patterns. The dendrogram at the top of the matrix indicates the degree of similarity between samples. b Multiple dimensional scaling based on the 36K oligo array human platform comparing Relapse free (Red color) and relapse (Green color)

Fig. 2

Supervised gene clustering and canonical pathway analysis. Heat map of the 349 genes, identified by Student’s t test (P = 0.001) comparing relapse and relapse-free patients

Fig. 3

Unsupervised gene cluster analysis. a Sixteen genes among Mammaprint panel set differentially expressed among relapse (brown) and relapse-free (red) patients. b Thirteen genes among Oncotype DX assay panel set differentially expressed among relapse (brown) and relapse-free (red) patients. c Five genes selected from the 299 genes by Complete Leave-One-Out Cross Validation (LOOCV) model as best predictors of diagnostic outcome. Red dots under the cluster indicates relapse free and brown dots indicates relapse group

Fig. 4

Ingenuity pathway analysis. Forty-six canonical pathways significant at the nominal 0.001 level of the unpaired Student’s t test. The P value for each pathway is indicated by the bar and is expressed as –1 times the log of the P value. The line represents the ratio of the number of genes in a given pathway that meet the cutoff criteria divided by the total number of genes that make up that pathway

Fig. 5

Real-time PCR analysis of frozen tumor specimens of relapse-free versus relapse patients. Two cohorts of patients were included in the validation group, and their tumors were subjected to confirmatory real-time PCR. Data are presented as average of mean of triplicate wells after normalization to GAPDH

Fig. 6

IHC analysis of paraffin-embedded tumor specimens of relapse-free versus relapse patients. a Representative data (×400 magnification) from nine patients with relapse-free survival and eight patients with relapse are presented. Human tonsil was stained as positive control. b Cell counts are presented as percent positive cells of tumor-infiltrating cells counted in five fields and averaged using a ×400 magnification