The diverse outcomes of children with NBL-NA have been largely unexplained. Our study suggests for the first time that infiltrating inflammatory cells, especially TAMs, may contribute to this diversity. We demonstrate that TAMs are more prevalent in tumors of children with metastatic rather than locoregional neuroblastoma. Furthermore, we show that expression of inflammation-related genes is higher in tumors of children diagnosed at age ≥ 18 months and that a subset of these genes representing TAMs is associated with an extremely poor outcome in this group. Including expression of both inflammatory and tumor cell genes in a 14-gene signature enables prediction of disease progression for the first time in the clinically indistinguishable group of patients diagnosed at age ≥ 18 months with metastatic NBL-NA. The novel finding that five inflammation-related genes contribute to 25% of the accuracy of the 14-gene model emphasizes the role of inflammation in neuroblastoma and uncovers previously unrecognized potential targets for therapy. This 14-gene expression scoring model, which was validated in two independent cohorts of patients, has clinical applicability and may be of used for managing high-risk patients.
In recent years, the concept of inflammatory cells in the tumor microenvironment as critical participants in tumor progression has gained acceptance.1,32
A tumor-infiltrating macrophage and T-cell signature (CD68high
) has been reported to predict PFS and OS in patients with breast cancer.4
In our study, IL6R
expression was found to be highly correlated with expression of CD14
(macrophage marker) and CD33
(myeloid marker), and their expression, along with that of IL10
(M2 polarization), contributed to the accuracy of our model in predicting disease progression. This novel finding provides a validated clinical context for the recently established role of TAMs and bone marrow mesenchymal stem cells in promoting neuroblastoma growth via activation of the IL6
Melanomas and myelomas, similar to neuroblastomas, have also been shown to co-opt their tumor microenvironment cells to produce IL6
, leading to STAT3 activation and promotion of tumor growth.30,31,33,34
Studies using immunocompetent mouse cancer models have demonstrated that antibody responses against transformed cells could lead to recruitment and polarization of macrophages, leading to production of cytokines such as IL6
, and IL4
that in turn stimulate tumor growth and angiogenesis.35,36
In the current study, using a highly specific and sensitive TLDA assay, expression of genes related to the humoral immune system was observed in children diagnosed at age ≥ 18 months but did not contribute to the final predictive model. Additional studies are needed to elucidate the role of antibody response in neuroblastoma and its relation to recruitment and polarization of TAMs.
Genes related to tumor cells contributed most to the accuracy of the 14-gene NBL-NA signature and included NTRK2
, which binds brain-derived neurotrophic factor and plays an important role in the survival and differentiation of neuroblastoma cells.25,26,37,38
The association of high NTRK2
expression with aggressive behavior in metastatic NBL-NA was previously reported by our group21
and is further supported in this study. Our present work also reveals a novel association between expression of IL6R
. Although additional mechanistic studies are required to understand interactions between these two pathways, our data point to the role of a pro-tumor inflammatory microenvironment in enabling a highly aggressive neuroblastoma phenotype.
Several gene expression and genomic studies of neuroblastoma tumors obtained at diagnosis have previously reported associations with patient outcome.20,39–43
However, these studies analyzed groups of patients who were heterogeneous with respect to MYCN
gene amplification status, clinical stage, and age at diagnosis. Segmental genomic alterations identified in high-risk NBL-NA tumors have not been predictive of outcome in this high-risk group but have had clinical utility in children with intermediate-risk neuroblastoma.20,41,44–48
Gene expression–based models built using neuroblastoma samples from clinically heterogeneous groups of patients lack predictive accuracy in those diagnosed at age ≥ 18 months with metastatic NBL-NA.42,43
Similarly, our previously reported 55-gene NBL-NA–specific microarray signature21
was not predictive of outcome for patients with MYCN
-amplified tumors, which represent a distinct molecular subgroup of neuroblastomas (unpublished data).49
Overall, these findings suggest that prognostic studies in neuroblastomas should focus on well-defined molecular and clinical subgroups (eg, using MYCN
status). Our current finding also highlights the importance of assessing the neuroblastoma tumor microenvironment in prognostic studies.
Our current study defines a clinically applicable 14-gene expression signature that identifies two subsets of patients with different PFS. Children with high-risk tumor-progression scores uniformly had a poor outcome, with 8% to 20% PFS at 5 years after diagnosis, whereas those with low-risk scores had 47% to 57% PFS. It is possible that the addition of treatment response evaluations such as imaging with 123
I-metaiodobenzylguanidine and quantification of bone marrow disease with polymerase chain reaction assays will further improve risk classification, especially for patients with a low-risk tumor-progression score.50,51
In summary, our study reports the first evidence of a role for intratumor inflammation in metastatic neuroblastomas and provides a validated prognostic signature for children with metastatic NBL-NA. The increase in expression of inflammation-related genes in children age ≥ 18 months with poor outcome allows the identification of a subgroup of patients at extremely high risk who may benefit from treatments targeting the tumor microenvironment along with tumor cells. The recent success of therapies directed at tumor-associated immune system cells in adult cancers52–54
suggests opportunities for their application in children with neuroblastoma.