Kinetics of Blast Clearance in Cerebrospinal Fluid (CSF) Detected on Flow Cytometric Immunophenotyping (FCM), but Negative By Cytopathology in Newly Diagnosed Acute Lymphoblastic Leukemia Patients

Background: Flow cytometric immunophenotyping (FCM) detects two-fold higher CSF disease. Limited data on kinetics of CNS disease detected in this manner. Our study aimed to identify and follow course of CSF FCM+ in newly diagnosed ALL. Serial CSF FCM examinations performed to detect and see persistence of blasts. Identify risk factors for CSF involvement any correlation with post-induction measurable residual disease (PI-MRD).

Methods: Enrolled 125 patients newly diagnosed treatment naïve ALL patients from April 2022 to December 2023, Ethics approval and informed consent. CNS status assessed CSF cytopathology and FCM. Patients categorized as CNS1, CNS2, and CNS3 based on St Jude’s criteria. CNS1 patients further divided into Cyto^-FCM⁺ and Cyto^-FCM - groups. Additional CSF FCM samples collected D+15 and D+28. All FCM analysis performed on BD FACS Canto II flow cytometer and BD FACS Diva v8.0.3 used for analysis. Patients received augmented BFM or adult protocol. Intrathecal Mtx given after baseline CSF and administered on D+15 and D+28 of induction.

Statistical analysis- Qualitative variables assessed using Chi-square test or Fisher’s exact test. Quantitative variables assessed using ANOVA with post-hoc Bonferroni or Kruskal-Wallis test, p-value<0.05 was considered statistically significant.

Results: total 125 patients, 104 B-ALL, 16 T-ALL, and 5 MPAL, male-to-female ratio 3:1. Median age 16 years (1-57). CNS signs or symptoms present in 17 patients (13.6%) they had CNS2 or CNS3 status. Of remaining 108 patients, 22 (20.4%) were Cyto^-FCM⁺, and 86 (79.6%) were Cyto^-FCM-

Three patients in Cyto^-FCM⁺ group died before D+15. Of remaining 19 patients repeat CSF, FCM detected persistent blasts in 2 (10.5%) while 17 (89.5%) showed blast clearance. The 2 patients with persistent CNS blasts cleared blasts D+15 and D+28.

Cyto^-FCM- group vs CNS2/3 group significantly lower number of NCI high-risk patients (69.8% vs 100%). Cyto^-FCM⁺ group significant hepatomegaly compared to Cyto^-FCM- group (72.7% vs 46.5%). CNS2/3 and Cyto^-FCM⁺ group higher median total leucocyte counts (TLC ) compared to Cyto^-FCM^-- and Higher LDH (589 vs 651 vs 349.5 IU/L, p-0.012 and 0.02). Receiver operator characteristic curve analysis showed TLC >6.3X10^9/L and LDH more than 314IU/L could predict Cyto^-FCM⁺ with a sensitivity of 84.6% and 87.2% (p=0.0005 and 0.0006) respectively. Lowest end-of-induction MRD positivity was seen in Cyto^-FCM^- group.

Conclusions: FCM detects CSF higher involvement in newly diagnosed ALL patients. However, ~90% patients cleared CSF by standard intrathecal methotrexate by D+15 and all patients cleared disease by D+28 without extra intrathecal therapy. Longterm effect impacts needs evaluation.