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Page 10 of 12 Schaafsma et al. J Cancer Metastasis Treat 2021;7:34 https://dx.doi.org/10.20517/2394-4722.2021.72

reported that certain immune characteristics are associated with patient survival irrespective of MYCN
[40]
amplification status . B cell infiltration could thus be used as a prognostic marker in neuroblastoma in
addition to commonly utilized prognostic indications such as age, stage and MYCN amplification status.
Consistently, we observed significant associations between B cell infiltration and prognosis when adjusting
for clinical variables and MYCN status.

Although our study provides important insights into immune infiltration in neuroblastoma, a few
limitations should be noted. First, all of our findings are based on gene expression data, which might not
always recapitulate protein expression. Protein-based approaches such as immunohistochemistry should
corroborate our findings of high B cell infiltration in patients with a better prognosis. Evaluation of the
presence of tertiary lymphoid structures adjacent to neuroblastoma tumors should be performed as well.
Second, although we attempted to evaluate the association between immune cell infiltration and common
chromosomal abnormalities in neuroblastoma, notably Chr1p deletion, Chr11q deletion, and Chr17q gain,
only few studies contained this information. Additional studies with available information on chromosomal
deletions and gains should be performed to validate our findings. Lastly, our prognostic analyses were all
performed retrospectively and prospective studies should evaluate the exact value of B cell as a prognostic
biomarker in neuroblastoma.

In conclusion, we have provided a comprehensive evaluation of immune cell infiltration in neuroblastoma
using gene expression data. The infiltration of naïve B cells, NK cells, and CD8+ T cells is associated with
better prognosis in neuroblastoma among which naïve B cells are the most consistent indicator of
prognosis. Based on further analyses, we propose a critical role for B cells in the neuroblastoma TME. The
presence of high B cell infiltration is associated with an immune-infiltrated TME and could be used as a
prognostic biomarker to predict recurrence-free and overall survival independently of currently utilized
prognostic variables.

DECLARATIONS
Acknowledgments
We would like to thank all members of the Cheng lab for their suggestions and critical feedback.

Authors’ contributions
Conceived and supervised the project: Cheng C
Performed computational analyses: Schaafsma E, Jiang C, Cheng C
Wrote the manuscript: Schaafsma E
Interpreted the results: Schaafsma E, Jiang C, Cheng C
Critically reviewed the content: Schaafsma E, Jiang C, Cheng C
Read and approved the final manuscript: Cheng C, Jiang C, Cheng C

Availability of data and materials
All data utilized in this study are publicly available. See Supplementary Table 1 and Methods for data
sources.

Financial support and sponsorship
This work is supported by the Cancer Prevention Research Institute of Texas (CPRIT) (RR180061 to Cheng
C), the National Cancer Institute of the National Institutes of Health (1R21CA227996 to Cheng C), and the
T32 training grant of the National Institutes of Health (T32 AI007363 to Schaafsma E). Cheng C is a CPRIT
Scholar in Cancer Research.