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<figure><img src="image_42.png" /><figcaption>**Figure 1.** ETMRs regardless of *C19MC* amplification show high molecular similarity

a-b, *t*-SNE clustering using either DNA methylation (a) or mRNA expression data (b). Colors represent different tumor entities classified as described previously⁷. c, Violin plot showing log2 transformed expression of *C19MC* miRNAs (n=56) in ETMRs and other tissues. P-values were calculated using two-sided Mann-Whitney U tests (***= P<0.0005). Boxplots show the median ± interquartile range, whiskers extend to 1.5x the interquartile range, violin plots depict kernel density estimates and represent the density distribution.</figcaption></figure>
<figure><img src="image_27.png" /><figcaption>**Figure 1.** ETMRs regardless of *C19MC* amplification show high molecular similarity

a-b, *t*-SNE clustering using either DNA methylation (a) or mRNA expression data (b). Colors represent different tumor entities classified as described previously⁷. c, Violin plot showing log2 transformed expression of *C19MC* miRNAs (n=56) in ETMRs and other tissues. P-values were calculated using two-sided Mann-Whitney U tests (***= P<0.0005). Boxplots show the median ± interquartile range, whiskers extend to 1.5x the interquartile range, violin plots depict kernel density estimates and represent the density distribution.</figcaption></figure>
<figure><img src="image_43.png" /><figcaption>**Figure 2.** ETMRs without *C19MC* amplification recurrently harbor miRNA related aberrations a, Oncoplot showing somatic events occurring in ETMRs. b, Overview of identified *DICER1* mutations. Alternating blue colors represent exons, yellow bars represent domains and pins represent the different aberrations found. c, Quantification of miRNA processing using the median ratio between 3p and 5p miRNAs (n=375), each bar representing one tumor. P-values were calculated using a one-sided Mann-Whitney *U* test (*** P<0.0005).</figcaption></figure>
<figure><img src="image_28.png" /><figcaption>**Figure 2.** ETMRs without *C19MC* amplification recurrently harbor miRNA related aberrations *a*, Oncoplot showing somatic events occurring in ETMRs. *b*, Overview of identified *DICER1* mutations. Alternating blue colors represent exons, yellow bars represent domains and pins represent the different aberrations found. *c*, Quantification of miRNA processing using the median ratio between 3p and 5p miRNAs (n=375), each bar representing one tumor. P-values were calculated using a one-sided Mann-Whitney *U* test (*** P<0.0005).</figcaption></figure>
<figure><img src="image_44.png" /><figcaption>**Figure 3.** Primary/relapse comparison reveals poor conservation of SNVs but high conservation of SVs</figcaption></figure>

a, Graph depicting allele frequencies of combined SNVs found between four primary tumors and matched initial relapses. Boxes represent SNVs that are gained, conserved and lost upon relapse. b, Analysis of mutational signatures of primary and relapsed tumors based on previously defined mutational signatures¹³,¹⁴. c, Radial plot depicting log2 fold changes of the somatic SNV burden between primary tumor (PRIM) and subsequent relapses (REC1 and REC2) colored by exposures shown in b. Number of mutations in primary tumor was set to 1 for visualization purposes. d, Venn diagram showing the overlap of breakpoints between a primary tumor and matched relapses. e, Circular representation of the genome of SVs and CNAs in a primary tumor with two matched recurrences shown in d. The outer rim represents, from outer to inner, the CNAs found in the primary tumor, the first recurrence and second recurrence. The middle part represents the different SVs found between primary tumors and recurrences. Chromosome 19 and X have been enlarged.
<figure><img src="image_29.png" /><figcaption>**Figure 3.** Primary/relapse comparison reveals poor conservation of SNVs but high conservation of SVs</figcaption></figure>

a, Graph depicting allele frequencies of combined SNVs found between four primary tumors and matched initial relapses. Boxes represent SNVs that are gained, conserved and lost upon relapse. b, Analysis of mutational signatures of primary and relapsed tumors based on previously defined mutational signatures¹³,¹⁴. c, Radial plot depicting log2 fold changes of the somatic SNV burden between primary tumor (PRIM) and subsequent relapses (REC1 and REC2) colored by exposures shown in b. Number of mutations in primary tumor was set to 1 for visualization purposes. d, Venn diagram showing the overlap of breakpoints between a primary tumor and matched relapses. e, Circular representation of the genome of SVs and CNAs in a primary tumor with two matched recurrences shown in d. The outer rim represents, from outer to inner, the CNAs found in the primary tumor, the first recurrence and second recurrence. The middle part represents the different SVs found between primary tumors and recurrences. Chromosome 19 and X have been enlarged.
<figure><img src="image_45.png" /><figcaption>**Figure 4.** Breakpoint context reveals a possible role for R-loops in initiating ETMRs

a,Schematic representation of GO-term enrichment. Circles represent GO-terms, sizes enrichment and colors groups based on similarity scores (Co-occurrence Association Score >0.05) (Supplementary Table 3). b, Enrichment in DRIP signal around *C19MC* (fold enrichment over input). c, Density of ETMR breakpoints (n=2301) overlapping DRIP-peaks (left; n=16002) and RLFS (right; n=85534) compared to random regions of the same size. P-values were calculated using a two-sided Chi-square test (***= P<0.0005). d, Enrichment of breakpoints overlapping RLFS compared to 10000 randomly generated sets of regions of the same size for ETMRs and other entities²². Boxes show the range (median, first and third quartile) of BH adjusted P-values calculated using one-sided Fisher's exact tests, whiskers extend to upper and lower limits of the data (90% and 10% respectively). e, Immunohistochemistry pictures of full slides (left) and magnifications (right) for ETMRs (n=5) and a representative case of WNT (n=5) and Group4 MB (n=5). f, Immunohistochemistry of WT and *DCR-KO* cell lines stained for DAPI (blue), S9.6 (red) and y-H2AX (green). g, Quantification of signals shown in f, in mean signal per cell (n=255 WT cells, n=227 *DCR-KO* cells), normalized to DAPI signal. P-values were calculated using a two-sided Mann-Whitney *U* test (***= P<0.0005). Boxes show median, first and third quartile and whiskers extending to 1.5x the interquartile range. h, Dot blot of DNA-RNA hybrids extracted from WT and *DCR-KO* cells, ssDNA was used as loading control.</figcaption></figure>
<figure><img src="image_30.png" /><figcaption>**Figure 4.** Breakpoint context reveals a possible role for R-loops in initiating ETMRs

a, Schematic representation of GO-term enrichment. Circles represent GO-terms, sizes enrichment and colors groups based on similarity scores (Co-occurrence Association Score >0.05) (Supplementary Table 3). b, Enrichment in DRIP signal around *C19MC* (fold enrichment over input). c, Density of ETMR breakpoints (n=2301) overlapping DRIP-peaks (left; n=16002) and RLFS (right; n=85534) compared to random regions of the same size. P-values were calculated using a two-sided Chi-square test (***= P<0.0005). d, Enrichment of breakpoints overlapping RLFS compared to 10000 randomly generated sets of regions of the same size for ETMRs and other entities²². Boxes show the range (median, first and third quartile) of BH adjusted P-values calculated using one-sided Fisher's exact tests, whiskers extend to upper and lower limits of the data (90% and 10% respectively). e, Immunohistochemistry pictures of full slides (left) and magnifications (right) for ETMRs (n=5) and a representative case of WNT (n=5) and Group4 MB (n=5). f, Immunohistochemistry of WT and *DCR-KO* cell lines stained for DAPI (blue), S9.6 (red) and y-H2AX (green). g, Quantification of signals shown in f, in mean signal per cell (n=255 WT cells, n=227 *DCR-KO* cells), normalized to DAPI signal. P-values were calculated using a two-sided Mann-Whitney *U* test (***= P<0.0005). Boxes show median, first and third quartile and whiskers extending to 1.5x the interquartile range. h, Dot blot of DNA-RNA hybrids extracted from WT and *DCR-KO* cells, ssDNA was used as loading control.</figcaption></figure>
<figure><img src="image_46.png" /><figcaption>**Figure 5.** ETMR cells are sensitive to combination therapy with PARP and TOP1 inhibitors
a, Dose response curves of ETMR cells treated with either Pamiparib or Veliparib, Topotecan and a combination of both drugs, P-values were calculated using two-way ANOVA. Error bars denote mean ± s.e.m. b, Calculation of synergy using the Chou-Talay method of drug treatments shown in a. c, Immunohistochemistry of ETMR cells stained for y-H2AX and S9.6. Cells were treated using IC50 concentrations of every drug or combination. d, Quantification of signal shown in c, in mean signal per cell (n=59 DMSO treated, n=158 Pamiparib treated, n=88 Topotecan treated, n=20 Combination treated cells), normalized by the total DAPI signal in the cell. P-values were calculated using two-sided Mann Whitney *U* tests (***= P<0.0005).</figcaption></figure>
<figure><img src="image_31.png" /><figcaption>**Figure 5.** ETMR cells are sensitive to combination therapy with PARP and TOP1 inhibitors
a, Dose response curves of ETMR cells treated with either Pamiparib or Veliparib, Topotecan and a combination of both drugs, P-values were calculated using two-way ANOVA. Error bars denote mean ± s.e.m. b, Calculation of synergy using the Chou-Talay method of drug treatments shown in a. c, Immunohistochemistry of ETMR cells stained for y-H2AX and S9.6. Cells were treated using IC50 concentrations of every drug or combination. d, Quantification of signal shown in c, in mean signal per cell (n=59 DMSO treated, n=158 Pamiparib treated, n=88 Topotecan treated, n=20 Combination treated cells), normalized by the total DAPI signal in the cell. P-values were calculated using two-sided Mann Whitney *U* tests (***= P<0.0005).</figcaption></figure>
<figure><img src="image_47.png" /><figcaption>Figure 6. Hallmarks of ETMR</figcaption></figure>

Schematic summary of ETMR characteristics
<figure><img src="image_32.png" /><figcaption>Figure 6. Hallmarks of ETMR</figcaption></figure>

Schematic summary of ETMR characteristics