Low Flow Cytometry Events in Detecting CNS Involvement in Pediatric BCP-ALL: NGS Validation and Prognostic Significance.

Central nervous system (CNS) status in pediatric acute lymphoblastic leukemia (ALL) is a key prognostic factor that makes precise detection critical. Flow cytometry (FCM) offers a higher detection rate than conventional cytology (CC); however, results are considered negative with < 10 FCM events, limiting its sensitivity. This study analyzed the cerebrospinal fluid from 173 pediatric patients with ALL using both CC and FCM, characterizing immunoglobulin heavy chain rearrangement clonotypes using next-generation sequencing (NGS). The sensitivities of CC and FCM were similar (P = 0.824) when the standard FCM positivity threshold was used. Receiver operating characteristic analysis based on NGS showed ≥ 3 events as the optimal FCM positivity cutoff, significantly improving sensitivity over CC (P = 0.005). Among patients treated according to the Taiwan Pediatric Oncology Group 2013 ALL protocol, FCM positivity correlated with lower event-free survival (EFS) and higher cumulative incidence of CNS relapse (CIRCNS). Patients with hyperdiploidy who were CC-negative/FCM-positive had worse EFS and CIRCNS when receiving less intensive CNS-directed treatments. The three-event cutoff for FCM positivity enhances the detection of CNS involvement in pediatric ALL and, when combined with CC, identifies patients at a higher risk of CNS relapse, highlighting the potential need for treatment intensification.