The prognostic significance of KLRB1 and its further association with immune cells in breast cancer

Abstract

Background: Killer cell lectin-like receptor B1 (KLRB1) is an important member of the natural killer cell gene family. This study explored the potential value of KLRB1 as a breast cancer (BC) biomarker and its close association with the tumor immune microenvironment during the development of BC.

Methods: We examined the differential expression of KLRB1 in pan-cancer. Clinical and RNA-Seq data from BC samples were evaluated in The Cancer Genome Atlas (TCGA) and validated in Gene Expression Omnibus (GEO) datasets and by immunohistochemistry (IHC) staining. The relationship between KLRB1 and clinical parameters was explored through Chi-square tests. The diagnostic value of KLRB1 was evaluated using a receiver operating characteristic (ROC) curve. Survival analysis was tested by Kaplan-Meier curves to demonstrate the relationship between KLRB1 and survival. Univariable and multivariate cox regression analyses were carried out as well. The analysis of immune infiltration level and gene set enrichment analysis (GSEA) were conducted to examine KLRB1's mechanism during the progression of BC. We used the Tumor Immune Estimation Resource (TIMER), the Cancer Single-cell Expression Map (CancerSCEM) database, the Tumor Immune Single-cell Hub (TISCH) database, and the Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT) method to explore KLRB1's association with immune infiltration level and different quantitative distribution of immune cells. The relevant signaling pathways in BC associated with KLRB1 were identified using GSEA.

Results: The expression of KLRB1 was downregulated across the majority of cancers including BC. The lower KLRB1 expression group exhibited shorter relapse free survival (RFS) and overall survival (OS). IHC staining showed that KLRB1 staining was weaker in breast tumor tissues than in paratumors. Additionally, GSEA identified several pathway items distinctly enriched in BC. KLRB1 expression level was also positively related to the infiltrating number of immune cells in BC. Moreover, the CancerSCEM and TISCH databases as well as the CIBERSORT method demonstrated the close relationship between KLRB1 and immune cells, particularly macrophages.

Conclusion: Low KLRB1 expression was considered an independent prognostic biomarker and played an important role in the tumor immune microenvironment of BC patients.

Keywords: Biomarker; Breast cancer; Killer cell lectin-like receptor B1 (KLRB1); Macrophages; Prognosis; Tumor immune microenvironment.

Figure 1. (A) Expression difference between normal and tumor tissues, meaningful cancer types including bladder urothelial carcinoma, breast invasive carcinoma, colon adenocarcinoma, rectum adenocarcinoma, head and neck squamous cell carcinoma, liver hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, thyroid carcinoma, uterine corpus endometrial carcinoma, kidney renal clear cell carcinoma, and kidney renal papillary cell carcinoma. P-value significant codes: 0 ≤ ∗∗∗ < 0.001 ≤ ∗∗ < 0.01 ≤ ∗ < 0.05 ≤ . < 0.1. The survival analysis of cancers of bladder urothelial carcinoma (B), lung carcinoma (C), liver hepatocellular carcinoma (D), thyroid carcinoma (E), colorectal cancer (F), breast cancer (J), renal cancer (H), head and neck carcinoma (I) and uterine corpus endometrial carcinoma (J).

Figure 2. KLRB1 expression in breast tumors.

The expression of KLRB1 is lower in tumors (A). Differences in KLRB1 expression were shown in clinical stage (p = 0.0130) (B), molecular subtype (p < 0.0001) (C), histological type (p < 0.0001) (D), T classification (p = 0.0010) (E), N classification (p = 0.0210) (F), M classification (G), sample type (H), age (p < 0.0001) (I), gender (p = 0.0083) (J), margin status (K), menopause status (p < 0.0001) (L), radiation therapy (p = 0.0008) (M), neoadjuvant treatment (N), targeted molecular therapy (p = 0.0120) (O) and vital status (p = 0.0095) (P).

Figure 3. The ROC curve of KLRB1 in breast cancer.

ROC curve for KLRB1 expression in breast cancer and normal tissues (A) (AUG: 0.727). Subgroup analyses: Stage I (B) (AUG: 0.685), Stage II (C) (AUG: 0.746), Stage III (D) (AUG: 0.702), and Stage IV (E) (AUG: 0.801). Different stages of breast cancer also showed certain diagnostic value. Abbreviations: AUC, area under the curve; ROC, receiver operating characteristic.

Figure 4. Kaplan-Meier curves of OS in breast cancer according to KLRB1 expression in breast cancer tissues.

Overall survival analysis (A) and subgroup analyses of histological type (B and C), ER (D and E), PR (F and G), HER-2 (H and I), and molecular subtype (J, K, and L). Low KLRB1 expression showed a relationship with poor overall survival (p < 0.0001).

Figure 5. Kaplan-Meier curves of RFS in breast cancer according to KLRB1 expression in breast cancer tissues.

Relapse free survival analysis (A) and subgroup analyses of histological type (B and C), ER (D and E), PR (F and G), HER-2 (F and G), and molecular subtype (J, K, and L). Low KLRB1 expression showed a relationship with poor relapse free survival (p = 0.0046).

Figure 6. Multivariate Cox analysis of KLRB1 expression and other clinical pathological factors in OS (A) and RFS (B).

Figure 7. The relevance of KLRB1 expression in the GEO database.

KLRB1 expression in normal breast tissues was higher than in breast cancer tissues (p = 0.0140).

Figure 8. The quantitative distribution of immune cells in breast cancer datasets GSE128673 (A) and GSE143423 (B).

The results are based on the Cancer Single-cell Expression Map database (https://ngdc.cncb.ac.cn/cancerscem/index).

Figure 9. The different expression of KLRB1 between immune cells and stromal cells.

KLRB1 possesses higher expression in immune cells than in stromal cells. The results are based on the TISCH database (http://tisch.comp-genomics.org/home/).

Figure 10. The expression levels of KLRB1 are negatively correlated with tumor purity (A) in basal-like (B), HER2-enriched (C), and luminal (D) breast cancer.

The KLRB1 expression level had significant positive correlations with infiltrating levels of B cells (p = 2.11e − 30, r = 0.355), CD8 + T cells (p = 9.45e − 62, r = 0.494), CD4 + T cells (p = 9.88e − 70, r = 0.527), macrophages (p = 1.56e − 03, r = 0.101), neutrophils (p = 2.71e − 36, r = 0.392), and dendritic Cells (p = 4.46e − 63, r = 0.506) in breast cancer.

Figure 11. The association between 22 distinct leukocyte subsets and high and low expression groups of KLRB1 shown by heatmap.

The high expression group of KLRB1 showed the higher expression of T cells CD8, T cells CD4 memory resting, and macrophages M1, and lower expression of macrophages M0 and macrophages M2.

Figure 12. The higher expression could be seen in high KLRB1 group in the subsets of B cells memory (p < 0.001), T cells CD8 (p < 0.001), T cells CD4 memory resting (p < 0.001), T cells CD4 memory activated (p < 0.001), T cells gamma delta (p < 0.001), macrophages M1 (p < 0.001), mast cells activated (p < 0.001) and neutrophils (p < 0.001), and lower expression in macrophages M0 (p < 0.001) and macrophages M2 (p < 0.001).

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Figure 13. The correlation matrix between each leukocyte subset shown by correlation heatmap in breast cancer.

The colored squares are used to display the correlation coefficients (red, positive Spearman’s rho; blue, negative Spearman’s rho).

Figure 14. Kaplan-Meier curves in breast cancer according to the high and low expression of distinct leukocyte subsets.

The survival analysis showed significantly higher expression of macrophages M1 (A) as well as T cells gamma delta (B), and lower expression of macrophages M2 (C) and monocytes (D) was associated with higher survival rate in breast cancer.

Figure 15. (A–F) Enrichment plots from GSEA.

The GSEA results indicated that epithelial mesenchymal transition, inflammatory response, kras signaling up, tnfa signaling via NFKB, IL6 JAK STAT3 signaling, and coagulation pathways were differentially enriched in high and low KLRB1 expression groups.

Figure 16. Immunohistochemistry staining for KLRB1.

The expression of KLRB1 in breast cancer cell was decreased. (A, B) IHC staining of paratumor breast tissues ((A) × 40, (B) × 100); (C, D) IHC staining of breast cancer tissues ((C) × 40, (D) × 100).