Figure 5.
Multimodal detection and characterization of CLTC::SYK fusions. (A) Exemplary butterfly plot of TLC-NGS data, uncovering the CLTC::SYK fusion in case 12. Proximity ligation products between the target gene (SYK) and rearrangement partner (CLTC) are depicted in green. The shade of green indicates the coverage. Strand directions are indicated by arrows. Details of TLC-NGS methodology are available in a prior publication by Allahyar et al.22 (B) Illustration of the CLTC::SYK fusion, which leads to a transcript joining CLTC exon 31 to SYK exon 6, as demonstrated by RNA sequencing. (C) Photomicrograph showing separation of red and green signals in lesional cells of the tumor of case 10 using SYK break-apart FISH. The green signal represents the FITC-labeled probes (which bind proximal of SYK), whereas the red signal represents the Cy3-labeled probes (which bind distal of SYK). (D) Sanger sequence demonstrating the fusion of CLTC intron 31 to SYK intron 5 in case 9. Sanger sequencing results for all patients with CLTC::SYK fusions are depicted in supplemental Figure 3. (E) Exemplary photomicrographs of the tumor of case 2, demonstrating expression of SYK, p-SYK, cyclin D1, and p-S6 (a marker of mTOR activation). In addition, rare p-ERK+ cells can be observed, whereas tumor cells were negative for p-Akt despite adequate positive external controls (endometrial cancer with PIK3CAH1047R mutation; SignalSlide Akt Family IHC Control from Cell Signaling). The SignalSlide Akt Family IHC Control is shown in the inlet. P-ERK expression of all tumors with CLTC::SYK fusions is depicted in supplemental Figure 2. (F) Exemplary multispectral immunofluorescence image of the tumor of case 12, demonstrating SYK and p-SYK expression by CD163+ histiocytes. (G) Unsupervised hierarchical clustering of 29 XG family lesions and 18 common or atypical dermatofibromas (DF; included as controls) using the methylation data of the 20 000 most variable probes. In the heat map, columns represent samples, and rows represent probes. The level of DNA methylation is indicated by beta values and depicted using a color scale. A beta value of 1 corresponds to a fully methylated CpG site; a value of 0 to an unmethylated site. Above the heat map, the clinical and/or molecular subgroup is shown for each sample; below the heat map, case numbers of patients described in this study are depicted. FITC, fluorescein isothiocyanate; IHC, immunohistochemistry. Panel B illustration made with the St Jude ProteinPaint web application.

Multimodal detection and characterization of CLTC::SYK fusions. (A) Exemplary butterfly plot of TLC-NGS data, uncovering the CLTC::SYK fusion in case 12. Proximity ligation products between the target gene (SYK) and rearrangement partner (CLTC) are depicted in green. The shade of green indicates the coverage. Strand directions are indicated by arrows. Details of TLC-NGS methodology are available in a prior publication by Allahyar et al.22 (B) Illustration of the CLTC::SYK fusion, which leads to a transcript joining CLTC exon 31 to SYK exon 6, as demonstrated by RNA sequencing. (C) Photomicrograph showing separation of red and green signals in lesional cells of the tumor of case 10 using SYK break-apart FISH. The green signal represents the FITC-labeled probes (which bind proximal of SYK), whereas the red signal represents the Cy3-labeled probes (which bind distal of SYK). (D) Sanger sequence demonstrating the fusion of CLTC intron 31 to SYK intron 5 in case 9. Sanger sequencing results for all patients with CLTC::SYK fusions are depicted in supplemental Figure 3. (E) Exemplary photomicrographs of the tumor of case 2, demonstrating expression of SYK, p-SYK, cyclin D1, and p-S6 (a marker of mTOR activation). In addition, rare p-ERK+ cells can be observed, whereas tumor cells were negative for p-Akt despite adequate positive external controls (endometrial cancer with PIK3CAH1047R mutation; SignalSlide Akt Family IHC Control from Cell Signaling). The SignalSlide Akt Family IHC Control is shown in the inlet. P-ERK expression of all tumors with CLTC::SYK fusions is depicted in supplemental Figure 2. (F) Exemplary multispectral immunofluorescence image of the tumor of case 12, demonstrating SYK and p-SYK expression by CD163+ histiocytes. (G) Unsupervised hierarchical clustering of 29 XG family lesions and 18 common or atypical dermatofibromas (DF; included as controls) using the methylation data of the 20 000 most variable probes. In the heat map, columns represent samples, and rows represent probes. The level of DNA methylation is indicated by beta values and depicted using a color scale. A beta value of 1 corresponds to a fully methylated CpG site; a value of 0 to an unmethylated site. Above the heat map, the clinical and/or molecular subgroup is shown for each sample; below the heat map, case numbers of patients described in this study are depicted. FITC, fluorescein isothiocyanate; IHC, immunohistochemistry. Panel B illustration made with the St Jude ProteinPaint web application.

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