Clinical significance of tumor-associated inflammatory cells in metastatic neuroblastoma

Abstract

Purpose: Children diagnosed at age ≥ 18 months with metastatic MYCN-nonamplified neuroblastoma (NBL-NA) are at high risk for disease relapse, whereas those diagnosed at age < 18 months are nearly always cured. In this study, we investigated the hypothesis that expression of genes related to tumor-associated inflammatory cells correlates with the observed differences in survival by age at diagnosis and contributes to a prognostic signature.

Methods: Tumor-associated macrophages (TAMs) in localized and metastatic neuroblastomas (n = 71) were assessed by immunohistochemistry. Expression of 44 genes representing tumor and inflammatory cells was quantified in 133 metastatic NBL-NAs to assess age-dependent expression and to develop a logistic regression model to provide low- and high-risk scores for predicting progression-free survival (PFS). Tumors from high-risk patients enrolled onto two additional studies (n = 91) served as independent validation cohorts.

Results: Metastatic neuroblastomas had higher infiltration of TAMs than locoregional tumors, and metastatic tumors diagnosed in patients at age ≥ 18 months had higher expression of inflammation-related genes than those in patients diagnosed at age < 18 months. Expression of genes representing TAMs (CD33/CD16/IL6R/IL10/FCGR3) contributed to 25% of the accuracy of a novel 14-gene tumor classification score. PFS at 5 years for children diagnosed at age ≥ 18 months with NBL-NA with a low- versus high-risk score was 47% versus 12%, 57% versus 8%, and 50% versus 20% in three independent clinical trials, respectively.

Conclusion: These data suggest that interactions between tumor and inflammatory cells may contribute to the clinical metastatic neuroblastoma phenotype, improve prognostication, and reveal novel therapeutic targets.

Fig 1.

Evidence of tumor-associated macrophages and inflammation in neuroblastoma. (A) Representative immunohistochemical analyses of staining of CD163 (brown staining) and AIF1 (red staining) in primary tumor samples from a patient with stage 1 tumor (left panel) lacking any infiltrating macrophages and a patient with metastatic disease (right panel) with extensive infiltration of CD163+ macrophages. (B) Average scores for the presence of CD163+ infiltrating macrophages reveals significant infiltration in tumor samples of patients with metastatic disease compared with those with locoregional tumors. Patients with stage 4S tumors, who are known to undergo spontaneous regression, had CD163+ macrophages comparable to those with locoregional tumors (Bonferroni adjusted P < .017 considered significant). (C) Heatmap of gene expression levels of inflammation-related genes displayed along increasing age at diagnosis of 133 children with metastatic MYCN-nonamplified neuroblastoma. Heatmap colors reflect fold-change value of individual tumor relative to the average expression level of inflammation-related genes in children diagnosed at age < 18 months. Occurrences of an event and of death are indicated by black vertical bars above the color-coded bar for age at diagnosis.

Fig 2.

Progression-free survival (PFS) for patients in the training and validation cohorts with metastatic MYCN-nonamplified neuroblastoma (NBL-NA) 14-gene signature low- and high-risk scores. The cutoff value used to categorize patients into signature-based high- and low-risk score groups depended on the median score obtained in the Children's Cancer Group (CCG) cohort. The high-risk score group had prediction scores higher than the median score, whereas the low-risk score group had prediction scores lower than the median. The graphs show Kaplan-Meier estimates of PFS for patients with NBL-NA according to 14-gene signature risk classification. PFS estimates using the leave-one-out cross-validation signature classification for (A) the entire CCG cohort (n = 133) and (B) CCG patients diagnosed at age ≥ 18 months (clinically defined high-risk group; n = 94). The classification model developed using the CCG samples was then used to identify signature-based groups for patients age ≥ 18 months treated on (C) German Society for Pediatric Oncology-Hematology high-risk protocols (n = 39) and (D) Children's Oncology Group high-risk protocols (n = 52). All P values are based on log-rank test.

Fig 3.

Inflammation- and tumor-related gene-gene correlations. (A) Heatmap of the Spearman rank correlation matrix of the 14 genes in the metastatic MYCN-nonamplified neuroblastoma (NBL-NA) signature. Pairwise rank correlation analyses were performed for all 14 genes and age at diagnosis. The patterns of correlation using samples from Children's Cancer Group (CCG) patients diagnosed at age ≥ 18 months were similar to the pairwise rank correlations obtained using samples from the German Society for Pediatric Oncology-Hematology and Children's Oncology Group validation cohorts (Data Supplement). Red represents positive rank correlation level above zero (white color) for a given gene pair, and blue represents negative rank correlation level. The inflammation-related genes (FCGR3/CD16, CD33, CD14, IL6R, IL10) show high levels of correlation across all cohorts. NTRK2 has the strongest correlation of any tumor cell–related gene with inflammation-related genes. (B) Expression of inflammation-related genes is predominantly present in neuroblastoma tumors and not on cell lines. Normalized expression (ΔCT) values of four of the five inflammation-related genes (CD14, FCGR3/CD16, IL10, IL6R) in the CCG tumors (n = 133) were compared with six neuroblastoma MYCN nonamplified cell lines (CHLA-15, CHLA-20, CHLA-255, CHLA-42, CHLA-90, LAN-6). Data on the cell lines were generated on a TaqMan low-density array (Life Technologies, Carlsbad, CA) card that did not include CD33 probes. On average, there was 309-, 271-, seven-, and five-fold higher expression of CD14, FCGR3/CD16, IL10, and IL6R in tumors than in cell lines, respectively, suggesting that these genes are primarily expressed by tumor-associated inflammatory cells. (C) Scatter diagram of expression of CD14, a macrophage marker, and IL6R reveals a high correlation in patients with metastatic NBL-NA. (D) Similarly, NTRK2, a tumor cell–related gene, shows moderate correlation with expression of IL6R, an inflammation-related gene. Gray, patients diagnosed at age < 18 months with metastatic NBL-NA; blue, disease-free patients diagnosed at age ≥ 18 months; gold, disease progression in patients diagnosed at age ≥ 18 months.