Trial Information

Potential Role of CD9 and Implication of Motility Process in Pathogenesis of TEL/ALM1-positive ALL Relapses (LAL TEL/ALM1 and CD9).

1. Assess of the impact of CD9 expression level on motility assays (migration and
adhesion) We have initiated motility assays (fibronectin adhesion experiments and
CXCL12 chemoattracted migration tests with modified Boyden chamber technique) using the
CD9 positive TEL/AML1-positive cell line REH and the CD9 negative cell line RAJI (wild
or transfected with CD9 cDNA). Data will be analyzed in combination with blocking
antibodies and chemical antagonist according to the level of CD9 (transcript and
protein) and of CXCR4. Protein quantifications will be performed by flow cytometry and
Western Blot. Interactions will be explored by confocal microscopy and biological
pathways by immunoblot.

Adhesion results will be validated on patient samples of B-ALL.

2. Post-transcriptional regulation of CD9 in TEL/AML1-positive ALL To identify miRNAs that
are potentially deregulated in TEL/AML1-positive acute lymphoblastic leukaemia and
especially to screen for CD9 -targeted miRNAs, we will use a TaqMan ®MicroRNA Arrays
approach allowing the simultaneous measurement of about 760 human miRNA.

Small RNA will be extracted from bone marrow samples of twenty childhood B-ALL to screen
miRNAs which are differentially expressed between CD9-positive and CD9-negative ALL and
further compared with miRNAs which were predicted to target CD9 in databases. Validation of
the selection will be performed by single Q-PCR for selected miRNAs using a novel cohort of
ten bone marrow samples. Transfection assays and luciferase assays will be further realized
to confirm that the differential miRNAs really target and affect CD9 expression .