Figure 3. C19MC detection is diagnostic for GLUT-1⁺ IH.

(A) Photograph of a child (NICH 264) clinically diagnosed with NICH who underwent surgical excision. (B) qRT-PCR analysis of the C19MC cluster was performed on cryopreserved tissue from the lesion in A as well as 3 other NICHs (NICH 121, 169, and 364) and one GLUT-1⁺ IH (IH 344). x-axis labels represent individual C19MC miRNA qRT-PCR assays. y-axis (linear scale) values represent ΔCt between the global mean of all tested miRNAs - assayed miRNA. NICH 169 and 264, but not 121 and 364, contained levels of C19MC miRNAs similar to the proliferative IH 344. All miRNAs below the limit of detection were assigned a ΔCt value of –14. (C) Paraffin sections were prepared from blocks preserved at the time of surgery and stained for GLUT-1. For NICH 121 (top row, DAB staining), GLUT-1 positivity is represented in brown. For NICH 169 and 264 (middle and bottom rows), GLUT-1 positivity is represented by green fluorescence. White arrows indicate GLUT-1^– endothelial cells; magenta arrows indicate GLUT-1⁺ endothelial cells. Only the C19MC-expressing NICHs 169 and 264 expressed endothelial GLUT-1. Scale bar: 100 μm. (D) Individual qRT-PCR assays demonstrate GLUT-1 and miR-517c-3p expression in both NICH 169 and 264 (purple bars), which was absent in NICH 121 (black bars, background GLUT-1 is due to presence of erythrocytes). The y axis represents relative mRNA or miRNA compared with levels of GAPDH or RNU48, respectively. Individual data points represent experimental duplicate samples prepared from the same lesion, and error bars represent standard deviations.