Flow Cytometric Leukemic Blasts Detection in Cerebrospinal Fluid of Children with Acute Lymphoblastic Leukemia

Background: Central nervous system (CNS)-directed treatment has reduced risk of CNS relapse for childhood acute lymphoblastic leukemia (ALL), which accounts for 30-40% of initial relapses. Compared with CNS-negative patients, patients with CNS leukemia (>5 leukocytes/µL cerebrospinal fluid (CSF) and lymphoblasts) suffer from more CNS+ relapses. Conventional cytology (CC) is specific (>95%), but nonsensitive (<50%), since leukocytes in CSF decay within few hours after lumbar puncture.

Method: We prospectively assessed centralised multi-parameter flow cytometry (FCM) of fixated CSF versus local CC in Nordic/Baltic childhood ALL. Diagnostic samples from 172 children aged 0-18 years with de novo and eight children with relapsed ALL were investigated in 297 CSF samples from 180 patients. Kinetics of disappearance of leukemic cells in the CSF was evaluated until day 15. Antibody-combinations reflected the immunophenotype of leukemic blasts in bone marrow.

Result: Of 172 newly diagnosed patients, 51 (30%) had CSF involvement by FCM, while CC was positive in 16 patients (9%) (p<0.001). CSF involvement was detected by both FCM and CC in four of eight patients with relapse (50%). Among newly diagnosed patients, samples positive by FCM and CC had higher leukemic blast count compared to samples positive by FCM only (medians: 0.545 (range: 0.005-4.801) versus 0.016 (range: 0.003-1.38) leukemic blasts/µL; p<0.001). Among newly diagnosed patients with samples positive by FCM and CC, the CSF blast count was related to the CSF leukocyte count (r_s=0.82; p=0.001). Compared to newly diagnosed patients who were FCM-negative, those with FCM-positivity had higher WBC (median: 37 versus 9 x 10⁹/L; p<0.001), were younger (medians: 3 versus 5 years, p=0.04), and more often had T-cell ALL (12/51 (24%) versus (6/121 (5%), p<0.001). Five (16%) of 31 newly diagnosed patients with FCM detected blasts at diagnosis and available data at day 15 still had CSF leukemic blasts on day 15. So far the two patients who later developed CNS and/or bone marrow relapse were positive by flow (1.38 and 0.178 blasts/µL), but CNS negative by CC or had CSF leukocyte count <5/µL and lymphoblasts on CC, respectively.

Conclusion: Leukemic blasts are present in spinal fluid of one third of newly diagnosed ALL. CSF involvement is associated with other higher risk characteristics. The prognostic value of these findings awaits prospective evaluation.