Quantification of P2RY8-CRLF2 genomic breakpoints and CRLF2 transcripts. (A) Relation between abundance of genomic fusion and CRLF2 expression in 56 P2RY8-CRLF2–positive ALL cases. Genomic DNA was used for the fusion (triangles; empty symbols, cases with P2RY8-CRLF2 in a minor subclone [< 20% of leukemia population]; filled symbols, cases with P2RY8-CRLF2 in a major clone [≥ 20% of leukemia population]); and mRNA for CRLF2 transcript quantification (squares). Black symbols indicate cases in remission; and red, relapse cases. (Left) Y-axis: normalized CRLF2 levels plotted relative to CRLF2 expression in peripheral blood MNC. (Right) Amounts of genomic fusion in percentage of the leukemia population. X-axis: ALL cases ordered from the lowest to highest CRLF2 expression. A dashed horizontal line has been inserted at the 50-fold CRLF2 overexpression defined by the lowest expression in cases with the fusion in a major population of the leukemia clone and no expression in all other genetic ALL subtypes. The black vertical line separates cases with low (left) and high (right) CRLF2 expression. (B) Quantification of CRLF2 expression by real-time PCR: comparison of 2 methods. (Left) Y-axis: ΔCt values of CRLF2 expression using the Applied Biosystems kit and the criteria for high expression of ΔCt ≤ 8.0. (Right) Fold changes of CRLF2 expression, as in panel A. X-axis: ALL cases ordered from the lowest to highest percentage of CRLF2 expression as in panel A. Two horizontal lines were inserted to show the respective limits for overexpression by each method; dashed, the 50-fold overexpression as in panel A; dotted, CRLF2 overexpression at ΔCt ≤ 8.0. (C) Relation between abundance of genomic fusion and CRLF2 expression by the inventoried assays for CRLF2 and EEF2. The dotted horizontal line indicates the CLRF2 expression at ΔCt 8.0 as in panel B. ALL cases ordered from the lowest to highest CRLF2 expression as in panel B. The black vertical line separates cases with low (left) and high (right) CRLF2 expression.