Prospective validation of VNN2 by FCM confirms its association with MRD. (A) FCM evaluation of VNN2 in diagnostic samples from 1069 B-cell ALL patients enrolled in the AIEOP-BFM 2009 study. (B) Classification of 72 BFM 2009 VNN2⁺ patients based on cytogenetic information and FCM data. Cytogenetic data were available for 72 of 103 patients identified by FCM. (C) VNN2 expression by FCM identifies TCF3-HLF–rearranged samples. Green dots indicate TCF3-HLF–rearranged samples (patient and PDX), and pink dots indicate TCF3-PBX1–rearranged PDX samples. Arrows indicate matched patient-PDX sample or matched xenograft samples at diagnosis-relapse. Asterisks (*) indicate matched diagnosis-relapsed samples. TCF3-HLF rearranged samples scored positive for VNN2 by FCM (>10%). (D) Analysis of VNN2 in matched samples from 9 patients taken at diagnosis of ALL (day 0) and at day 15 by FCM. (E) Comparison of matched diagnosis (black circles) and relapse (gray rhombus) pairs indicated conservation of VNN2 positivity; in some cases, an increase in VNN2 from diagnosis to relapse could be observed. FCM was used to evaluate VNN2 of matched diagnosis and first relapse of 22 patients with ALL. (F) Ex vivo drug response profiling of 10 VNN2⁺ ALL samples (red circles) compared with 8 VNN2⁻ SR cases (blue circles). Patient samples were tested for dexamethasone (DEX), mitoxantrone (MITOX), bortezomib (BORTEZ), vincristine (VINCR), doxorubicin (DOX), and idarubicin (IDA). **P < .05. BAL, biphenotypic acute leukemia according to EGIL classification; B-ALL, B-cell ALL; dx, diagnosis; FMO, fluorescence-minus-one; IC50, 50% inhibitory concentration; MPAL, mixed phenotype ALL, according to World Health Organization classification²³ ; rz, relapse; SE, standard error.