MicroRNA-181a as novel liquid biopsy marker of central nervous system involvement in pediatric acute lymphoblastic leukemia

Bálint Egyed,Judit Müller,Tamás Visnovitz,András Gézsi,Gábor T Kovács,Péter Lőrincz,Dániel J Erdélyi,Andrea Rzepiel,Judit C Sági,Ágnes F Semsei,Nóra Kutszegi,Csaba Szalai,Marianna Zombori

doi:10.1186/s12967-020-02415-8

Abstract

BackgroundRefractory central nervous system (CNS) involvement is among the major causes of therapy failure in childhood acute leukemia. Applying contemporary diagnostic methods, CNS disease is often underdiagnosed. To explore more sensitive and less invasive CNS status indicators, we examined microRNA (miR) expressions and extracellular vesicle (EV) characteristics.MethodsIn an acute lymphoblastic leukemia (ALL) discovery cohort, 47 miRs were screened using Custom TaqMan Advanced Low-Density Array gene expression cards. As a validation step, a candidate miR family was further scrutinized with TaqMan Advanced miRNA Assays on serial cerebrospinal fluid (CSF), bone marrow (BM) and peripheral blood samples with different acute leukemia subtypes. Furthermore, small EV-rich fractions were isolated from CSF and the samples were processed for immunoelectron microscopy with anti-CD63 and anti-CD81 antibodies, simultaneously.ResultsRegarding the discovery study, principal component analysis identified the role of miR-181-family (miR-181a-5p, miR-181b-5p, miR-181c-5p) in clustering CNS-positive (CNS+) and CNS-negative (CNS‒) CSF samples. We were able to validate miR-181a expression differences: it was about 52 times higher in CSF samples of CNS+ ALL patients compared to CNS‒ cases (n = 8 vs. n = 10, ΔFC = 52.30, p = 1.5E−4), and CNS+ precursor B cell subgroup also had ninefold higher miR-181a levels in their BM (p = 0.04). The sensitivity of CSF miR-181a measurement in ALL highly exceeded those of conventional cytospin in the initial diagnosis of CNS leukemia (90% vs. 54.5%). Pellet resulting from ultracentrifugation of CNS+ CSF samples of ALL patients showed atypical CD63−/CD81− small EVs in high density by immunoelectron microscopy.ConclusionsAfter validating in extensive cohorts, quantification of miR-181a or a specific EV subtype might provide novel tools to monitor CNS disease course and further adjust CNS-directed therapy in pediatric ALL.

Highlights

Refractory central nervous system (CNS) involvement is among the major causes of therapy failure in childhood acute leukemia
Prophylactic regimens against CNS leukemia has become a prerequisite of successful treatment: prior to the introduction of CNStargeted therapy in the 1960s, symptomatic meningeal leukemia developed in more than 50% of cases and CNS relapse rate was over 65% in childhood acute lymphoblastic leukemia (ALL) [3, 4]
We provide an analysis of miRs as putative biomarkers of pediatric acute leukemia stage and dynamics in the CNS compartment and a novel proposal of examining small extracellular vesicle (EV) in the cerebrospinal fluid (CSF) of CNS leukemia patients

Summary

Introduction

Refractory central nervous system (CNS) involvement is among the major causes of therapy failure in childhood acute leukemia. Patients with ALL who have a history of overt CNS involvement at the initial diagnosis are still susceptible to relapse with only 15–20% 5-year survival rates [5]. CNS disease in acute myeloid leukemia (AML) entails poor outcomes, and optimal treatment has not been established, mainly due to the rarity of this condition [6]. Due to the paucity of cells in the CSF, both methods yield a high proportion (more than 40%) of false-negative reports among patients with proven CNS infiltration by neuroimaging or autopsy [11]. A gold standard method for staging the CNS involvement in acute leukemia has not been established yet

Methods

Results

Discussion

Conclusion