Low intratumoral genetic neutrophil-to-lymphocyte ratio (NLR) is associated with favorable tumor immune microenvironment and with survival in triple negative breast cancer (TNBC)

Abstract

Patients with triple negative breast cancer (TNBC) have a poor prognosis. A novel prognostic biomarker may guide management by appropriately selecting patients for particular treatments. Peripheral blood neutrophil-to-lymphocyte ratio (NLR) was reported to associate with cancer progression, thus we hypothesized that intratumor genetic NLR will reflect tumor immune microenvironment (TIME) and breast cancer biology. The intratumoral genetic NLR previously defined as the ratio of CD66b (CEACAM8) and CD8 (CD8A) gene expressions was utilized to analyze total of 2,994 patients from METABRIC, TCGA, GSE21094, GSE22358, GSE25088, GSE32646, and GSE2603 cohorts. Intratumoral genetic NLR did not correlate with cancer stage nor clinical parameters of cancer cell proliferation such as Nottingham histological grade or MKI67 expression levels in neither the METABRIC or TCGA cohorts. Intratumoral genetic NLR-high breast cancer was not associated with pathologic complete response (pCR) after neoadjuvant chemotherapy in 5 independent cohorts with different regimens. Despite these results, intratumoral genetic NLR-high TNBC demonstrated worse disease-free, disease-specific, and overall survival. Intratumoral genetic NLR-low TNBC enriched multiple immune-related gene sets, was associated with higher favorable immune-related scores and with a favorable TIME, whereas no gene sets enriched to NLR-high TNBC. In conclusion, intratumoral genetic NLR-low TNBC was associated with favorable TIME and with better survival.

Keywords: GSEA; NLR; Neutrophil-to-lymphocyte ratio; TNBC; breast cancer; neoadjuvant chemotherapy; triple negative breast cancer; tumor immune microenvironment; xCell.

Figure 1

Intratumoral genetic neutrophil-to-lymphocyte ratio (NLR) does not vary by cancer stage. A. Box plot comparing intratumoral genetic NLR by stage in estrogen receptor (ER) positive/HER2 negative (ER+/HER2-) (METABRIC n=1001, Stage 1=363, Stage 2=568, Stage 3=62, Stage 4=8; TCGA n=576, Stage 1=115, Stage 2=319, Stage 3=133, Stage 4=9), HER2-positive (HER2+) (METABRIC n=171, Stage 1=47, Stage 2=96, Stage 3=27, Stage 4=1; TCGA n=191, Stage 1=19, Stage 2=117, Stage 3=52, Stage 4=3), and triple negative breast cancer (TNBC) (METABRIC n=217, Stage 1=62, Stage 2=130, Stage 3=25, Stage 4=0; TCGA n=159, Stage 1=29, Stage 2=100, Stage 3=25, Stage 4=2) subtypes. B. Box plot comparing intratumoral genetic NLR between primary breast cancers with metastasis (to bone or lung) and without metastasis in the GSE2603 cohort.

Figure 2

Intratumoral neutrophil-to-lymphocyte ratio (NLR) does not vary by clinical parameters indicative of cancer cell proliferation and cell death. A. Box plot of intratumoral genetic NLR by Nottingham histological grade in the METABRIC and TCGA cohorts. B. Box plot of MKI67 expression levels by intratumoral genetic NLR-low versus -high tumors in the METABRIC and TCGA cohorts. C. Box plot of cell death related gene expression levels by intratumoral genetic NLR-low versus -high tumors in the METABRIC and TCGA cohorts. Intratumoral genetic NLR-low and -high groups were divided by the median cutoff.

Figure 3

Intratumoral neutrophil-to-lymphocyte ratio (NLR)-high tumor was not associated with pathologic complete response (pCR) after neoadjuvant chemotherapy (NAC) in the GSE21094, GSE22358, GSE25066, and GSE32646 cohorts. The patients were divided into low and high groups using median cutoff of intratumoral genetic NLR.

Figure 4

Association between intratumoral neutrophil-to-lymphocyte ratio (NLR) levels and breast cancer patient survival in the METABRIC cohort. Kaplan-Meier survival curves comparing intratumoral genetic NLR high and low groups for disease-free survival (DFS), disease-specific survival (DSS), and overall survival (OS).

Figure 5

Gene set enrichment analysis (GSEA) and immune related scores by intratumoral genetic NLR-high versus -low triple negative breast cancer (TNBC) in the METABRIC and TCGA cohorts. A. Enrichment plots of immune related gene sets. False discovery rate (FDR) <0.25 was defined as statistically significant. B. Association between intratumoral genetic NLR levels and immune related scores in TNBC of TCGA cohort. NES, normalized enrichment score; FDR, false discovery rate; IFN-γ, interferon gamma; TIL, tumor infiltrating lymphocytes.

Figure 6

Association of intratumoral neutrophil-to-lymphocyte ratio (NLR) levels and infiltrating immune cells or the cytolytic activity score (CYT) in triple negative breast cancer (TNBC). A. Box plot of anti-cancer immune cells. B. Box plot of pro-cancer immune cells. C. Box plot of cytolytic activity (CYT) in the TCGA cohort. CD8+, CD8 positive T cell; CD4+, CD4 positive T cell; CYT, cytolytic activity score; DC, Dendritic cell; M1, macrophage M1; M2, macrophage M2; Th1, helper T cell type 1; Th2, helper T cell type 2; Treg, regulatory T cell.