1. Demographic and clinicopathologic characteristics
The study design is shown in Figure 1. A total of 113 eligible glioblastoma, IDH wild-type patients were identified in the TCGA database and selected in this study. In the TCGA dataset, there was no statistically significant difference in age, sex, whether radiotherapy or chemotherapy was performed, and MGMT promoter methylation status among patients in the TANs high group and low group (P > 0.05). Similarly, the CGGA RNA-seq database with 173 glioblastoma, IDH wild-type samples was used as a validation cohort. In CCGA dataset, there was statistically significant difference in chemotherapy among patients in the TANs high group and low group (P < 0.05). There were no statistically significant differences in age, sex, whether radiotherapy was performed, and MGMT promoter methylation status (P > 0.05)(Table 1).
2. Survival of patients and potential prognostic factors for OS
(1) TCGA dataset
In the TCGA dataset, clinical follow-up was available for 113 patients. The median survival time of patients in the TAN high group was 11.4 months, and was 15.1 months for patients in the TAN low group, and there was a statistically significant difference in overall survival between the groups (χ² = 6.0, P = 0.014; Figure 2A). In the TCGA dataset, univariate Cox analysis have shown that the infiltration of TANs (HR = 1.931, 95% CI: 1.139-3.274), radiotherapy (HR = 0.339, 95% CI: 0.187-0.616), and chemotherapy (HR = 0.610, 95% CI: 0.381-0.977) were factors that significantly influenced the prognosis of patients with glioblastoma, IDH wild-type(Figure 2G). Multivariate Cox regression showed that the infiltration of TANs (HR = 1.811, 95% CI: 1.048-3.128) and radiotherapy (HR = 0.381, 95% CI: 0.163-0.889) were independent factors influencing the prognosis of patients with glioblastoma, IDH wild-type.
(2) External Validation
In the CGGA dataset, follow-up details were available for 173 patients. The median survival time of patients in the TAN high group was 12.6 months, and was 15.8 months for patients in the TAN low group; there were statistically significant differences in overall survival between the two groups (χ² = 9.3, P = 0.002; Figure S1A). In the CGGA dataset, univariate Cox analysis revealed that the infiltration of TANs (HR = 1.799, 95% CI: 1.227-2.637) and chemotherapy (HR = 0. 419, 95% CI: 0.285-0.616) were factors influencing the prognosis of patients with glioblastoma, IDH wild-type (Figure S1G). Multivariate Cox regression showed that the level of tumor-associated neutrophil infiltration (HR = 1.572, 95% CI: 1.046-2.363) and chemotherapy (HR = 0.406, 95% CI: 0.264-0.625) were independent prognostic factors for OS of glioblastoma and IDH wild-type patients.
3. Stratification Analysis
In the TCGA dataset, stratification analysis was performed based on age, sex, chemotherapy, and MGMT promoter methylation status. Among 113 patients aged ≤ 60 years, the median survival time of patients in the TAN high group was 12.0 months, and was 16.9 months for patients in the TAN low group, and a statistically significant difference in overall survival was identified between the two groups (χ² = 14.6, P < 0.001). In females, the median survival time of patients in the TAN high group was 5.83 months, and was 15.33 months for patients in the TAN low group, and there was a statistically significant difference in overall survival between the two groups, stratified by patient sex (χ² = 8.9, P = 0.003). Among patients who received no chemotherapy, the median survival time of patients in TAN high group was 4.73 months, and that of patients in the TAN low group was 10.97 months, indicating a statistically significant difference in overall survival between the two groups stratified by chemotherapy status (χ² = 5, P = 0.03). In patients with MGMT promoter methylation, the median survival time of patients in the TAN high group was 11.4 months, and was 16.3 months for patients in the TAN low group, indicating a statistically significant difference in overall survival between the two groups stratified by MGMT promoter methylation status (χ² = 9.5, P = 0.002). According to the univariate analysis, there was an interaction between neutrophil infiltration content, patient sex, and MGMT promoter methylation status (Figure 3).
In the CGGA dataset, stratification analysis was performed based on patient age, sex, radiotherapy status, and MGMT promoter methylation status. Among 173 patients aged ≥ 56 years, the median survival time of patients in the TAN high group was 9.0 months, and was 13.3 months for patients in the TAN low group, indicating a statistically significant difference in overall survival between the two groups ( χ² = 10.7, P = 0.001). In male patients, the median overall survival time of in the TAN high group was 12.7 months, and was 15.8 months in the TAN low group (χ² = 8.1, P = 0.004). For patients receiving radiation therapy, the median survival time in the TAN high group was 13.1 months, and was 15.8 months for patients in the TAN low group, indicating a statistically significant difference in overall survival between the two groups subset by radiation therapy status (χ² = 6.3, P = 0.01). Among patients with MGMT promoter methylation, the median survival time was 12.6 months in the TAN high group and 18.2 months in the TAN low group, indicating a statistically significant difference in overall survival between the two groups subset by MGMT promoter methylation status (χ² = 4.4, P = 0.04). Based on the univariate analysis, there was an interaction between neutrophil infiltration, radiotherapy, and MGMT promoter methylation status (Figure S2).
4. Sensitivity coefficient analysis
In the TCGA dataset, after adjusting for patient age, sex, radiation, chemotherapy, and methylation of MGMT promoter, the RR = 1.507 and E-value = 2.38 were determined for death in the TAN high (Figure 4). In the CGGA dataset, the RR = 1.572 and E-value = 2.52 were determined for death in the TAN high group (Figure S3).
5. Assessment of functional molecules of tumor-promoting mechanisms of neutrophils and infiltration of TANs
The linear correlation between levels of ARG1, EGF, HGF, MMP9, PDGFB, S100A8, and S100A9 and the metric of infiltration of TANs was determined by correlation coefficients. Correlation coefficient analysis demonstrated that ARG1, PDGF, MMP9, S100A8, and S100A9 were linearly correlated with infiltration of TANs (Figure 5A-G). Compared to the TAN low group, the expression of ARG1, S100A8, and S100A9 was significantly increased in the TAN high group (Figure 5H-N).
6. Patient characteristics of the peripheral blood neutrophils cohort
In the peripheral blood neutrophils cohort, 143 patients with glioblastoma, IDH wild-type were included, and there were no statistically significant differences in age, sex, radiation, chemotherapy, or methylation of MGMT promoter between the peripheral blood neutrophil high and low groups before radiation (Table S1).
The correlation between peripheral blood neutrophils and survival before radiation was analyzed; 50 patients died at the end of follow-up, with a median survival time of 21.8 months in the peripheral blood neutrophil high group, and 13 patients died in the low group, with a median survival time of 39.4 months. The overall survival of patients in the high group was significantly shorter than that in low group (χ² = 4.9, P = 0.026; Figure 6A). In accordance with the univariate and multivariate Cox regression models: the level of peripheral blood neutrophils before radiation (HR = 2.073, 95% CI: 1.077-3.990) was an independent risk factor affecting the overall survival of patients with glioblastoma, IDH wild-type (Figure 6E).