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Schaafsma et al. J Cancer Metastasis Treat 2021;7:34 https://dx.doi.org/10.20517/2394-4722.2021.72 Page 9 of 12

Figure 6. Immune cell infiltration and chromosomal abnormalities. (A) Bar graph showing the log2 (fold-change) [log2(FC)] comparing
immune infiltration between tumors with normal MYCN copy number and tumors with MYCN amplifications. Each comparison was
evaluated in seven independent datasets (top to bottom for each immune cell type): Westerman, Wang, SEQC, Rajbhandari, Henrich,
Oberthuer, and Kocak. Significance calculated using Wilcoxon-rank sum tests. (B) Comparison of naïve B and NK cell infiltration in
patients with or without Chr17q and MYCN chromosomal rearrangements in the Wang dataset. Significance calculated using Wilcoxon-
rank sum tests. (C) Percentage of immune cell variance explained by 4 chromosomal rearrangements. Horizontal bars indicate the
standard deviation and solid dots indicate the mean variance explained of 100 iterations in which the order of variables was randomly
shuffled in each iteration. Each comparison was evaluated in 3 independent datasets (top to bottom for each chromosomal
rearrangement): Wang, Lastowska, and Henrich.

hypothesize that the small number of tumor infiltrating B cells might originate from these B cell-enriched
locations that might not always be captured during biopsies or tissue sections. This hypothesis is in line with
recent observations of B cells in other cancer types, where B cell follicles can reside at the tumor
margin [29-31] . The presence of these B cell structures is highly associated with survival and an effective anti-
tumor immune response [29-31] .

A number of B cell-related mechanisms are operational in the TME. Antigen presentation by B cells is
[36]
likely a major contributor to the reported positive association between B cell infiltration and patient
survival. For example, the occurrence of antigen-specific interactions between T cells and B cells in tumor B
cell structures promote CD8+ T cell cytotoxicity in the TME [37,38] . Another anti-tumor mechanism is the
secretion of tumor-specific antibodies that mediate opsonization, antibody-dependent cellular cytotoxicity
by NK cells, or promote tumor cell phagocytosis by macrophages and granulocytes . Lastly, the secretion
[39]
of cytokines, including IFNγ and IL-12, by B cells promotes further activation of anti-tumor CD8+ T cells
and NK cells .
[39]

We confirmed the findings of previous studies which showed that MYCN amplified neuroblastoma tumors
have significantly lower immune cell infiltration compared to patients without MYCN amplifications [9-12] .
When separating patients without and with MYCN amplifications, we still observed that naïve B cell
infiltration was associated with overall survival and RFS. This is consistent with a previous study that