Immunophenotypic Investigation of Infant Acute Lymphoblastic Leukemia.
Authors: Popov Alexander , Tsaur Grigory , Verzhbitskaya Tatiana , Streneva Olga , Shorikov Egor , Saveliev Leonid , Fechina Larisa 
Published: October 2019

Abstract
Abstract 2547
Acute lymphoblastic leukemia (ALL) in children less than 1 year old is the relatively rare disease with specific biological features and poor outcome. It is also characterized by high incidence of MLL gene rearrangements. Immunophenotype of infants' leukemia varies due to presence or absence of MLL-rearrangements.

Aim of the study.
description of immunophenotype in infant acute lymphoblastic leukemia.

Methods.
Totally 421 cases of pediatric acute leukemia (AL) were studied. 81 patients (39 boys and 42 girls) aged from 5 days to 11 months were included in the study group. Their data was compared to 332 cases of acute leukemia in older children. Tumor cells immunophenotyping was performed by 6–8-color flow cytometry. Detection of various types of MLL-gene rearrangements was done by simultaneous application of chromosomal banding analysis, fluorescence in-situ hybridization, reverse-transcriptase polymerase chain reaction (PCR) and long-distance inverse PCR.

Results.
There were 54 (66.7%) ALL cases in the study group. ALL was found less frequently in infants than in older children (66.7% and 88.5% respectively, p<0.0001) while percentage of acute myeloid leukemia cases was higher in infants (27.2% and 10.0% respectively, p=0.0001). EGIL immunophenotypes distribution also differed between infants and older children. BI-ALL was the most common immunological ALL type in infant ALL (55.6% vs 3.3% in older age group, p<0.0001), while BII-ALL was notably less frequent compared with other age groups (33.3% and 77.1% respectively, p<0.0001). T-lineage ALL was also less frequent in infants (3.7% vs 14.3% in older age group) although difference did not achieve statistical significance (p=0.0536). Totally infant ALL were mainly presented by B-cell precursor ALL (BCP-ALL) – 51 patients (94.4%). Various types of MLL-rearrangements were found in 40 (74.1%) patients (pts) out of 54 infants ALL cases. Among them 21 pts (52.5%) carried MLL-AF4 fusion gene, 8 pts (20.0%) – MLL-MLLT1, 5 pts (12.5%) – MLL-MLLT3, 3 pts (7.5%) – MLL-EPS15, 1 pt (2.5%) – MLL-MLLT10, 1 pt (2.5%) – MLL-AFF3 and 1 pt (2.5%) had MLL-rearrangement with unidentified partner gene. Significant immunophenotypic differences were observed in patients with and without MLL gene rearrangements. Number of cases in those tumor cells expressed CD10, CD20, CD45, CD133, CD15, NG2 significantly varied between MLL-positive and MLL-negative groups (p=0.0001, p<0.0001, p=0.0008, p=0.0018, p=0.0306 and p<0.0001 correspondingly). NG2-positivity represented the highest overall correct prediction (OCP) rate for presence of MLL-rearrangements (95.5%). Diagnostic accuracy of CD20-negativity and CD45-positivity was slightly lower (87.5% and 86.3% respectively) while OCP for CD10-negativity (78.4%), CD133-positivity (75.0%) and CD15-positivity (66.7%) was not sufficient enough. Nevertheless CD10-positive BCP-ALL with MLL-rearrangements differed from CD10(+) cases in MLL-germline group. CD10 homogeneous expression was noted in 10 out of 11 MLL-germline patients and in 1 of 10 MLL-rearranged cases (p=0.0011). Although there were found no significant differences in CD22-positive patients' number, CD22(+)-cells percentage was significantly lower in MLL-positive cases (median 89.9%, range 25.2–99.7% and median 99.9%, range 96.0–99.9% respectively, p=0.0026). Thus CD20-negativity, CD10-negativity/low expression, high CD45, CD15, CD65 and NG2 expression, decreased CD22-expression are immunophenotypic signatures of MLL-rearranged infant ALL, although NG2 has the highest diagnostic efficacy. Interestingly there were no markers able to distinguish MLL-AF4-positive cases from patients carrying other types of MLL-rearrangements. Even NG2 expression intensity did not differ between these groups (p=0.2720). Except BCP-ALL pts, two cases of T-lineage ALL and one mature B-ALL (without other Burkitt lymphoma features) were found.

Conclusion.
Thus immunophenotype of ALL in children less than 1 year old differs significantly from patients of older age groups. Infants' B-cell precursor ALL immunophenotype varies greatly due to the presence of MLL gene rearrangements. Complex diagnostic immunophenotyping of infants' ALL allows predicting presence of MLL rearrangements and NG2 is the most applicable single marker.

Disclosures:
No relevant conflicts of interest to declare.