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inducing ligand (TRAIL) pathway and the death receptor pathway, are still active [112-117] . As a result, the tu-
mour is sensitive to natural killer (NK) and cytotoxic T-cell driven activation of these pathways, leading to
cell death. It has been shown that the presence of higher numbers of cytotoxic T-cells in the tumour micro-
environment correlated with better overall survival [118] .

Considering the sensitivity to NK and cytotoxic T cells, immunotherapy might be a promising therapeutic
intervention for EWS. NK clinical trials have started using immunotherapeutic strategies in the treatment of
EWS [119-122] . They involve treatment with donor NK cells after allogeneic haematopoietic cell transplantation,
or after receiving lymphodepleting chemotherapy and IL2, in some cases with a follow-up NK treatment 35
days after haematopoietic cell transplantation. In vitro results show that histone deacetylase inhibitors in-
crease expressions of NKG2D ligands on EWS cells, which can sensitize them for cytolysis via NK cells [123] .

Using primed or T cell receptor (TCR)-engineered T cells is a second approach for which EWSR1-ETS would
be a very selective target. However, preclinical studies haven’t been as successful in vivo as NK immunother-
apy, or lacked further follow up [124-127] . A limitation of this therapy is the dependency on MHC class I surface
expression, which is downregulated by EWS cells [128] .

Chimeric antigen receptor (CAR) T cells or NK cells can act independent of MHC class proteins and can
selectively be designed for all surface proteins, making it an interesting third approach. In leukaemia and
lymphomas, CAR-T cells were very successful in the clinic [129,130] . The main target investigated in EWS is the
glycolipid GD , which is expressed in most EWS tumours at varying levels and against which CAR-T cells
2
have been developed [131] . Preclinical studies targeting GD demonstrated a reduction in tumour volume and
2
a clinical trial with the latest CAR-T cells has been initiated [132] . As this therapy can be designed for every
surface expressed antigen one can imagine that other targets might be worth investigating such as the re-
[133]
cently identified surface protein LINGO1 .
A fourth approach of immunotherapy being investigated for clinical use are cancer vaccines. Higher num-
bers of CCL21-producing cells at the tumour site was correlated with an improved chemotherapy response
and overall survival: this might imply that attraction of dendritic cells and CCR7-positive T cells by a can-
cer vaccine or a dendritic cell-based cancer vaccine would help to induce long term protection. In addition,
cancer vaccines can change the suppressive immune environment by inducing a pro-inflammatory immune
response or inhibiting production of immune inhibitory proteins, such as transforming growth factor β1
(TGF-β1) [134-137] . The presence of a suppressive immune environment in EWS tumours is illustrated by low
the number of infiltrating immune cells, and T cell infiltration induced upregulation of HLA-G, an immune
inhibitory receptor [138] . In addition, in xenografts a high number of myeloid-derived suppressor cells were
detected in EWS tumours, leading to a reduction in CAR-T cell activity [139] . A phase IIb with a TGF-β1 in-
hibiting cancer vaccine is ongoing, but further insight in the immunosuppressive microenvironment of EWS
would be beneficial to design an immunotherapy which homes to the tumour site and has long-term tumour
specific cytotoxic activity.

CONCLUDING REMARKS
EWS has a fairly stable genome with a low number of somatic mutations. Aside from the characteristic
EWSR1-ETS translocation, only a limited number of recurrent mutations and copy number alterations
are found. Deeper insight into the underlying biology of the disease is slow to be obtained, due to its rar-
ity. However, owing to various collaborative efforts and use of state-of-the-art technologies in the field, the
needed headway is being made in order to develop novel therapeutics, including potential for immunothera-
peutic approaches. Current in vitro and in vivo models including zebrafish models are helpful in the process
for better understanding of the molecular genetics of these tumours and maybe more importantly, can act as
systems for exploring new therapeutic approaches.