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Figure 4. B cell infiltrated neuroblastoma associated with hot immune microenvironment. (A) Correlation between naïve B cell and
CD8+ T cell infiltration in the Oberthuer dataset. (B) Correlation matrix of immune cell infiltration estimates in the Oberthuer dataset.
(C) Correlation matrix of correlation coefficients between naïve B cell infiltration and 5 major immune cell types in 6 independent
neuroblastoma datasets. SCC: Spearman correlation coefficient.

High naïve B cells infiltration associated with enrichment of immune-related pathways
To further investigate characteristics of the TME of B cell-infiltrated neuroblastoma, we separated patients
based on low or high naïve B cell infiltration, using median naïve B cell infiltration as a separator. We
performed Gene Set Enrichment Analysis to assess which pathways were enriched in either patient group. A
distinct biological difference was observed, where pathways associated with cell proliferation were enriched
in patients with low B cell infiltration, whereas immune-related pathways were enriched in patients with
high B cell infiltration [Figure 5A]. For example, the Translation and Ribosome pathways were among the
most highly enriched pathways in patients with low B cell infiltration [Figure 5B], potentially reflecting
overall cell proliferation. Additional pathways related to cell proliferation, including eukaryotic translation
initiation, rRNA processing, DNA replication, and chromosome maintenance were also among the most
highly enriched pathways in patients with low B cell infiltration [Figure 5A]. Autoimmune thyroid disease
and IFNγ signaling were among the most highly enriched pathways in patients with high B cell infiltration
[Figure 5C]. Pathways related to transplant rejection were also enriched, including graft vs. host disease and
allograft rejection [Figure 5A], likely reflecting the presence of an ongoing immune response in B cell-
infiltrated tumors. In conclusion, low naïve B cell infiltration is associated with proliferative pathways
whereas high naïve B cell infiltration is associated with immune-related pathways.

Chromosomal abnormalities in relation to immune infiltration
Previous studies have investigated the relationship between specific copy number variations and immune
cell infiltration in neuroblastoma. Several studies have suggested that neuroblastoma tumors with MYCN
amplifications are poorly infiltrated [9-12] . We indeed confirmed the negative relationship between MYCN
amplifications and immune cell infiltration in seven independent datasets [Figure 6A]. Naïve B cells and NK
were the most consistent cell types associated with MYCN amplification status, showing significantly lower
immune infiltration in MYCN amplified samples in all seven datasets. In addition to MYCN amplifications,
other chromosomal abnormalities commonly occur in neuroblastoma. Only a small number of datasets
contained information on Chr1p, Chr11q, and Chr17q status, the most commonly altered chromosomal
abnormalities in neuroblastoma [2,32] . Since MYCN amplification is strongly associated with immune
infiltration, we separated samples based on MYCN status and each of the other chromosomal
rearrangements. Although we did observe some differences between naïve B and NK cell infiltration in
samples with and without Chr17q gains, MYCN amplification status was much more significantly
associated with the infiltration of these immune cells [Figure 6B]. TERT rearrangements and ATRX
mutations are also commonly observed in neuroblastoma [2,32] . While no difference in immune cell