VNN2 expression is associated with an increased risk for relapse in BCP ALL. (A) VNN2 mRNA expression by q-PCR correlates with detection of VNN2 at the cell surface by FCM. PDXs from patients included in the ALL-BFM-2000 study were used for correlation. The percentage of VNN2⁺ cells by FCM with respect to the fluorescence-minus-one control is plotted on the y-axis against the mRNA levels by q-PCR as logarithmic value of 2^-ΔCT (ΔCT = CT_VNN2 − CT_SDHA) on the x-axis. MRD-based risk stratification in ALL-BFM-2000 is indicated as SR, eMR, and HR. Patients with the translocation t(17;19) leading to TCF3-HLF are highlighted as TCF3-HLF⁺. (B) Five-year pEFS (upper part of the panel) and probability of cumulative incidence of relapse (pCIR; lower part of the panel) of 476 patients (test set) treated on the ALL-BFM 2000 study stratified according to VNN2 positivity by q-PCR. The cutoff for positivity was set to 1.13 ΔCT, as described in Materials and methods. (C-E) Five-year pEFS (upper part of the panel) and pCIR (lower part of the panel) of the same 476 patients in a subgroup analysis based on their risk stratification [SR (C), MR (D), and HR (E) for relapse] in the ALL-BFM-2000 study. A statistically significant difference was observed for pEFS and pCIR in VNN2⁺ vs VNN2⁻ patients in the MR group (P < .01). (F) Retrospective analysis of VNN2 expression by FCM on available archived samples from VNN2⁺ ALL patients based on q-PCR. Sample selection is described in supplemental Figure 1. Twenty-two of 40 positive samples by q-PCR were confirmed VNN2⁺ by FCM. Cutoff for VNN2 positivity by FCM > 10% (Materials and methods). pEFS of 22 VNN2⁺ patients based on FCM is plotted in comparison with pEFS of 615 VNN2⁻ patients based on q-PCR from the total retrospective cohort. P < .0001, log-rank test.