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Figure 5. Immune-related pathways enriched in neuroblastoma tumors with high naïve B cell infiltration. (A) Bar plot showing
normalized enrichment scores (NES) of pathways significantly enriched in tumors with low B cell infiltration (positive NES) and high B
cell infiltration (negative NES). (B) Gene Set Enrichment Analysis plots of the Translation and Ribosome pathways. (C) Gene Set
Enrichment Analysis plots of the Autoimmune thyroid disease and IFNγ signaling pathways.

infiltration based on TERT rearrangement status was observed, patients with ATRX mutations had
significantly lower levels of CD4+ T cell and higher levels of monocyte infiltration as compared to patients
without ATRX mutations [Supplementary Figure 2A-B].

In addition to assessing specific genotypic groups, we also assessed how much immune cell variation can be
explained by individual chromosomal rearrangements. Since the order of variables affects the percentage of
variation explained by each variable, we randomly shuffled the order of variables 100 times and calculated
the mean and standard deviation of the percentage of immune cell variance explained (see Methods). There
was considerable variation between datasets, but MYCN amplification status again showed the most
consistent results, especially in naïve B cell and NK cell infiltration [Figure 6C]. MYCN amplification status
explained approximately 10% of naïve B cell infiltration when considering four chromosomal
rearrangements in the model, while MYCN status accounted for approximately 25% of NK cell infiltration
in two out of three datasets [Figure 6C].

DISCUSSION
The presence of tumor infiltrating leukocytes is indicative of a host immune response to tumors and
infiltrating immune cells have been shown to be predictive of clinical outcomes for neuroblastoma
patients [9,35] . In our study, we show that several immune cell types are associated with recurrence-fee survival
(RFS) and overall survival, most notably naïve B cells, NK cells, and CD8+ T cells. We have expanded on
previous immune inference studies by utilizing a large number of gene expression datasets, as well as by
evaluating the association between prognosis and several immune cell types. We propose a previously
unappreciated role for naïve B cell abundance in neuroblastoma, which is highly associated with survival
and a hot TME.

Previous studies have suggested that only a small number of B cells infiltrate in neuroblastoma tumors [27,28] .
However, larger numbers of B cells might reside just outside the tumor. The presence of organized
[27]
lymphoid structures and B cell follicles at the edges of neuroblastoma tumors have been observed . We