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Page 4 of 12 Schaafsma et al. J Cancer Metastasis Treat 2021;7:34 https://dx.doi.org/10.20517/2394-4722.2021.72

RESULTS
Immune infiltration in neuroblastoma is associated with patient prognosis
To interrogate immune cell infiltration in neuroblastoma gene expression datasets, we inferred the
abundance of six common immune cell types, naïve B cells, memory B cells, CD4+ T cells, CD8+ T cells,
NK cells, and monocytes. This method has been well-established and validated in multiple studies [24,25] . We
first evaluated immune cell infiltration in the Oberthuer neuroblastoma gene expression dataset. We
observed that the infiltration of certain immune cells was positively associated with overall survival, while
the infiltration of other immune cells was negatively associated. High abundance of naïve B cells, memory B
cells, CD8+ T cells, and NK cells was significantly associated with longer overall survival [Figure 1A-D].
Conversely, high infiltration of CD4+ T cells was negatively associated with overall survival [Figure 1E]. The
infiltration of monocytes was not significantly associated with survival based on a Log-rank test
[Figure 1F]. Previous reports have suggested that few B cells infiltrate neuroblastoma tumors [27,28] and little is
known about their exact role in neuroblastoma. In addition, several studies have recently shown that B cells
are crucial in mounting an effective anti-tumor immune response [29-31] . In our study, naïve B cells were
highly significantly associated with survival as compared to the other major immune cell types, which
sparked our interest.

To validate our findings in the Oberthuer dataset, we collected several additional independent datasets
containing patient survival information [Supplementary Table 1] and inferred immune cell infiltration for
each dataset. We found a highly reproducible pattern where high infiltration of naïve B cells was
consistently associated with better overall survival [Figure 2A]. Increased infiltration of CD8+ T cells and
NK cells were also significantly associated with longer survival in more than half of the datasets
[Supplementary Figure 1A]. In addition to overall survival, recurrence-free survival was also significantly
longer in patients with high naïve B cell infiltration [Figure 2B]. In conclusion, these findings suggest that
naïve B cells are a reliable prognostic indicator in neuroblastoma.

Naïve B cells highly associated with survival independent of clinical variables
Naïve B cells were highly associated with patient prognosis in all evaluated datasets using univariate
analyses. However, several clinical variables are also known to be highly associated with overall patient
survival. The presence of MYCN amplifications (MYC-Gain) automatically classifies neuroblastoma as
high-risk [2,32] and patients with this amplification are treated more intensely to increase the probability of
overall survival. We thus stratified patients based on MYCN amplification status and evaluated the
association of naïve B cells with overall survival in MYC wild type and MYC-Gain patients [Figure 3A].
Patients exhibiting MYC-Gains indeed had much shorter overall survival, but patients with MYC-Gain and
high naïve B cell infiltration did live significantly longer in two out of three evaluated datasets and the last
datasets showed a trend of prolonged survival of patients with high naïve B cell infiltration [Figure 3A]. The
infiltration of naïve B cells in patients who did not have MYC-Gains was also significant in two out of three
datasets (P < 0.05) [Figure 3A].

In addition to MYCN amplification status, tumor stage and age are also important prognostic variables that
are considered during risk stratification [2,32] . Even after adjusting for these prognostic clinical variables and
MYCN amplification status, the infiltration of naïve B cells was still significantly associated with overall
survival in all independent datasets [Figure 3B]. Irrespective of tumor stage and patient age, the infiltration
of naïve B cells was consistently associated with longer patient survival. In addition, RFS was also
significantly longer in patients with high naïve B cell infiltration, irrespective of MYCN-amplification status
[Supplementary Figure 1B]. Adjustment of clinical variables and MYCN amplification status in multivariate
Coxph models showed that high naïve B cell infiltration is significantly associated with prolonged RFS
independent of adjusted variables [Supplementary Figure 1C]. In conclusion, the infiltration of naïve B cells