Genomic analysis of CRLF2 aberrations. (A) FISH analysis of DS-ALL expressing CRLF2 (i-ii) IGH@ CRLF2 translocation, patient DS-85: (i) Metaphase showing a positive result with the LSI IGH@ break-apart rearrangement probe (Abbott Molecular): normal chromosome 14 (yellow arrow) derived chromosome 14 (red arrow), derived X chromosome (green arrow). (ii) Interphase nucleus from the same patient hybridized with the homegrown CRLF2 probe showing a split signal pattern, 1R1G1F, confirming its involvement in the translocation (1 fusion signal [yellow arrow], 1 red signal [red arrow], and 1 green signal [green arrow]). (iii-iv) CRLF2 microdeletion, patient DS-82. (iii) Interphase nucleus hybridized with the IGH@ probe showing the normal 0R0G2F signal pattern confirming the presence of 2 normal copies of IGH@. (iv) Interphase nucleus from the same patient hybridized with the homegrown CRLF2 probe showing the deletion of the green portion of the probe (1 red signal [red arrow] and 1 fusion signal [yellow arrow]) denoting the presence of a centromeric interstitial deletion. (B) CRLF2 and P2RY8 expression in DS-ALL samples. Centered and normalized log basis 2 expression of CRLF2 and P2RY8 along DS-ALL samples in AIEOP dataset. Values for each individual case are represented by a color, with red representing deviation above the mean and blue representing deviation below the mean. The samples are sorted using SPIN. Pearson correlation between CRLF2 and P2RY8: −0.45 (P = .02). (C) Detection of the P2RY8-CRLF2 fusion transcript. (i) Schematic representation of the deletion breakpoint region at the telomeric end of chromosome X/Y with gene locations. The dashed lines represent the genomic deletion leading to the fusion of the first noncoding exon of P2RY8 and to the first (coding) exon of CRLF2. (iii) RT-PCR experiments on cDNA of DS patients. (Lanes 1-3) DS diagnostic ALL samples (DS93 and DS82 with FISH determined deletion and DS92 with FISH determined IgH@ translocation). (Lane 4) Blank. The 3 patient samples were positive for ABL amplification (not shown). Primer sets used are (A) P2RY8 F01/CRLF2 R01; (B) P2RY8 F01/ CRLF2 R02; (C) P2RY8 F01/CRLF2 R03 shown on the sequence on the left (ii). Chimeric transcripts are present in the first 2 lanes of each set. (ii) Nucleotide sequencing of the largest PCR fragment confirming the fusion transcript; vertical lines indicate exon boundaries; the arrowhead indicates the P2RY8/CRLF2 transcript junction. As seen, the fusion is just upstream to the ATG of CRLF2. Reference sequences are P2RY8-001 (ENST00000381297) and CRLF2-001 (ENST00000400841). Boxed sequence around the transcript junction is represented in the electropherogram on the right lower side (iv).