Small sizes and indolent evolutionary dynamics challenge the potential role of P2RY8-CRLF2-harboring clones as main relapse-driving force in childhood ALL

Abstract

The P2RY8-CRLF2 fusion defines a particular relapse-prone subset of childhood acute lymphoblastic leukemia (ALL) in Italian Association of Pediatric Hematology and Oncology Berlin-Frankfurt-Münster (AIEOP-BFM) 2000 protocols. To investigate whether and to what extent different clone sizes influence disease and relapse development, we quantified the genomic P2RY8-CRLF2 fusion product and correlated it with the corresponding CRLF2 expression levels in patients enrolled in the BFM-ALL 2000 protocol in Austria. Of 268 cases without recurrent chromosomal translocations and high hyperdiploidy, representing approximately 50% of all cases, 67 (25%) were P2RY8-CRLF2 positive. The respective clone sizes were ≥ 20% in 27% and < 20% in 73% of them. The cumulative incidence of relapse of the entire fusion-positive group was clone size independent and significantly higher than that of the fusion-negative group (35% ± 8% vs 13% ± 3%, P = .008) and primarily confined to the non-high-risk group. Of 22 P2RY8-CRLF2-positive diagnosis/relapse pairs, only 4/8 had the fusion-positive dominant clone conserved at relapse, whereas none of the original 14 fusion-positive small clones reappeared as the dominant relapse clone. We conclude that the majority of P2RY8-CRLF2-positive clones are small at diagnosis and virtually never generate a dominant relapse clone. Our findings therefore suggest that P2RY8-CRLF2-positive clones do not have the necessary proliferative or selective advantage to evolve into a disease-relevant relapse clone.

Figure 1. Identification of P2RY8-CRLF2 in childhood BCP-ALL cases

(A) PCR products of the genomic P2RY8-CRLF2 fusion of representative cases. Patient identification is indicated at the top of the gel: ad, nontemplate control; pB, peripheral blood MNCs; and SM, size marker. Left part of the gel (samples 360-903) shows PCR products from cases that were later defined as harboring the P2RY8-CRLF2 fusion in a major clone. Right part of the gel (samples 460-906) shows PCR products from ALL cases with P2RY8-CRLF2 in a minor subclone. (B) PCR for the fusion transcripts of corresponding cases. Note that sample 365 had no genomic PCR product but a distinct band for the transcript. (C) PCR products of the 6 cases with the newly discovered breakpoints upstream of CRLF2. A vertical line was inserted to indicate that different gels, run in parallel, from identical experiments were pasted together.

Figure 2. 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.

Figure 3. Clinical outcome of children with BCP-ALL enrolled in the BFM ALL 2000 protocol in Austria according to P2RY8-CRLF2

Kaplan-Meier estimates of 5-year pOS (left), pEFS (middle), and CIR (cumulative incidence; right) according to the presence of P2RY8-CRLF2 and its proportion in the leukemia of 248 screened non-DS cases (A,C) and of all 530 non-DS cases (B) enrolled in the treatment study. Analogous analyses of ALL cases treated according to the IR treatment arm (D-F).

Figure 4. Patterns of genomic P2RY8-CRLF2 and CRLF2 transcripts of fusion-positive ALL relapse cases

The 6 different patterns of clonal relation between initial diagnosis (D) and relapse (R) leukemia are depicted. (A) Genomic PCR products for P2RY8-CRLF2 in representative cases. Sample identification is indicated at the top of the figure: ad, nontemplate control; pB, peripheral blood MNCs; and SM, size marker. Cases 1, 3, and 737 harbor the fusion in a major clone at initial diagnosis, and it was either conserved (1), lost (3), or reduced to a minor subclone (737) at relapse. In cases 810, 564, and 243 with a fusion-positive minor subclone at initial diagnosis, the fusion was also either lost (810), preserved (243), or replaced by a different fusion (564) at relapse. Quantification of the genomic P2RY8-CRLF2 fusion (indicated in percentage of leukemia cells at the y-axis; B) and the corresponding CRLF2 transcript levels (fold changes of normalized expression of the samples over the expression in peripheral blood MNCs; C). Sample identification is given at the bottom of the graph.

Figure 5. Detection of the P2RY8-CRLF2 in neonatal blood spots

Amplification plots of the qRT-PCR of genomic P2RY8-CRLF2 using whole genome–amplified genomic Guthrie card DNA of 6 ALL cases: 400 (A), 737 (B), 802 (C), 833 (D), 841 (E), and 887 (F). Standard curves showing a quantitative range of 1 × 10⁻⁵ (blue) and PCR products of Guthrie card DNA from 4 aliquots in triplicates (red). PCR products for P2RY8-CRLF2 with a newly identified genomic breakpoint of case 873. (G) Amplification of the genomic breakpoint. Dilution series of genomic leukemia DNA. (Left) First round. (Right) Second round. The fusion can be detected at the single cell level at 1 × 10⁻⁵ (lane 5). Lane 1, size marker; lane 2, H₂O; lane 3, DNA mix from peripheral blood MNCs of healthy donors; and lanes 4 to 9, 10-log dilutions from 1 × 10⁻⁶ to 1 × 10⁻¹. Amplification of P2RY8-CRLF2 in native (H) and whole genome–amplified Guthrie card (I) DNA. Lane 1, size marker; lane 2, H₂O; lane 3, control DNA mix; and lanes 4 to 10, Guthrie cards DNA from a newborn who did not develop ALL later. a to i, PCR products from aliquots of the patients’ Guthrie card DNA.