Applicability and Prognostic Significance of Flow Cytometric Cerebrospinal Fluid Investigation in Children with Acute Lymphoblastic Leukemia

Conventionally central nervous system (CNS) involvement in acute lymphoblastic leukemia (ALL) is found by blasts detection in cerebrospinal fluid (CSF) via cytospin slides microscopy. It was shown earlier, that low levels of tumor cells, undetectable by cytomorphology (CM), could be found in several ALL cases using more sensitive techniques such as multicolor flow cytometry (MFC) (A. Popov et al, ASH-2011). Nevertheless prognostic value of these low levels of blasts in CSF is still unclear. The aim of present study was to evaluate the clinical significance of MFC application for initial diagnostics of leukemic cells in CSF of children with ALL.

Patients and methods. Since December 2008 till November 2014 in 155 consecutive children older than 1 year with ALL initial CNS involvement was investigated by CSF flow cytometry. Study group included 65 girls and 90 boys aged from 1 to 16 years (median age 4 years) treated according to ALL MB-2008 protocol. 134 patients (86.5%) suffered from various types of B-cell precursor ALL (BCP-ALL), while remaining 21 cases (23.5%) were T-lineage ALL. According to the protocol stratification system 59 patients belonged to standard risk group (SRG), 87 - to intermediate risk group (ImRG) and 9 - to high risk group. CSF lesion was studied in parallel by microscopy of cytospin slides and by MFC. CNS status was classified as CNS1 (no blast cells in CSF), CNS2 (<5 WBC/ul CSF with blast cells), or CNS3 (≥5 WBC/ul CSF with blast cells, or other signs of CNS involvement). Antibodies' combinations for cytometric CSF assessment were developed on the basis of leukemic cells immunophenotype defined by initial diagnostic bone marrow investigation. Treatment outcome was estimated by cumulative incidence of relapse (CIR). Median of follow up was 5 years.

Results. In 5 patients CNS3 status was defined. These patients were excluded from the further analysis due to obvious neuroleukemia signs. CNS2 was found in 23 of 150 remaining cases (15.3%) while 127 (84.7) had CNS1. Totally 53 out of 150 studied children (35.3%) had MFC-detectable CSF lesion. In 32 CNS1-patients leukemic blasts were found only by MFC. Absolute blast count in 1 ml in CSF samples, positive by both methods was significantly higher than in samples, positive only by MFC (median = 418, range 8-158171 and median = 34, range 5-2762 respectively, p<0.001). Thus MFC allows detecting tumor cells in CSF much more frequently (p<0.001) than conventional CM. This difference could be explained mainly by higher MFC sensitivity. Both CM and MFC presented results, valuable for outcome prediction. Children with CNS2 (n=23) had higher relapse incidence that patients with CNS1 (n=127): CIR 0.38(0.11) and 0.16(0.05) respectively, p=0.005. 53 MFC-positive patients displayed higher CIR compared to 97 MFC-negative ones: 0.26(0.06) and 0.15(0.05) respectively, p=0.019. Nevertheless MFC lacked prognostic impact in clinically relevant patients' groups. Among CNS1 cases the CIR difference between MFC-positive (n=32) and MFC-negative (n=95) groups was not significant: 0.19(0.07) and 0.15(0.05) respectively, p=0.148. SRG patients, who could be restratified according to the results of CSF inspection, also haven't displayed any difference in relapse incidence between 51 MFC-negative patients (CIR 0.18(0.07)) and 8 MFC-positive ones (CIR 0.27(0.17), p=0.313). In ImRG after exclusion of T-ALL cases cytometric CSF assessment also lacked its prognostic impact: MFC-negative group (n=33) had similar outcome to MFC-positive patients (n=33): CIR 0.08(0.06) and 0.17(0.07) respectively, p=0.239. In 37 initially MFC-positive cases follow-up CSF samples were also studied. Patients with fast clearance of CNS burden (n=24) displayed better outcome compared to children who remained MFC-positive (n=13), although these differences didn't achieve statistical significance: CIR 0.32(0.10) and 0.54(0.14) respectively, p=0.116.

Conclusions. Our data shows that MFC is able to find in CSF even small number of leukemic blasts, which are undetectable by conventional cytospin slides microscopy. Despite of this advantage of cytometric CSF inspection, the prognostic value of MFC data remains controversial. In contrast, larger tumor cells populations that could be detected in CSF also by cytomorpology are more strictly associated with high incidence of relapse. Nevertheless this data should be confirmed on larger patients' cohort in multicenter trial.