CD200/BTLA deletions in pediatric precursor B-cell acute lymphoblastic leukemia treated according to the EORTC-CLG 58951 protocol

Abstract

DNA copy number analysis has been instrumental for the identification of genetic alterations in B-cell precursor acute lymphoblastic leukemia. Notably, some of these genetic defects have been associated with poor treatment outcome and might be relevant for future risk stratification. In this study, we characterized recurrent deletions of CD200 and BTLA genes, mediated by recombination-activating genes, and used breakpoint-specific polymerase chain reaction assay to screen a cohort of 1154 cases of B-cell precursor acute lymphoblastic leukemia uniformly treated according to the EORTC-CLG 58951 protocol. CD200/BTLA deletions were identified in 56 of the patients (4.8%) and were associated with an inferior 8-year event free survival in this treatment protocol [70.2% ± 1.2% for patients with deletions versus 83.5% ± 6.4% for non-deleted cases (hazard ratio 2.02; 95% confidence interval 1.23-3.32; P=0.005)]. Genetically, CD200/BTLA deletions were strongly associated with ETV6-RUNX1-positive leukemias (P<0.0001), but were also identified in patients who did not have any genetic abnormality that is currently used for risk stratification. Within the latter population of patients, the presence of CD200/BTLA deletions was associated with inferior event-free survival and overall survival. Moreover, the multivariate Cox model indicated that these deletions had independent prognostic impact on event-free survival when adjusting for conventional risk criteria. All together, these findings further underscore the rationale for copy number profiling as an important tool for risk stratification in human B-cell precursor acute lymphoblastic leukemia. This trial was registered at www.ClinicalTrials.gov as #NCT00003728.

Figure 1.

Characterization of focal deletions of CD200/BTLA. CGH log₂ copy number ratio data shown as representation for (A) a mono-allelic deletion of CD200 and CD272 (BTLA) genes, (B) a mono-allelic deletion of CD200 gene and a bi-allelic deletion of CD272 (BTLA) in two different BCP-ALL cases and (C) a bi-allelic deletion of CD200 and CD272 (BTLA) in the REH cell line. (D) Quantitative reverse transcriptase-PCR analysis in eight B-ALL cell lines confirmed that deletion of CD200 and CD272 (BTLA) abolishes their expression in the REH cell line. The expression profiles of CD200 and CD272 (BTLA) are shown on a logarithmic scale in (E) three B-ALL cell lines and (F) BCP-ALL patients. The top panel shows the expression level of CD200 and CD272 (BTLA) in one wild-type and one deleted case and the box plots in the lower panel represent the mean fluorescence intensity of CD200 and CD272 (BTLA) in three deleted and 20 non-deleted BCP-ALL cases. Asterisks indicate samples used to generate the top panels.

Figure 2.

Genomic breakpoints of CD200/BTLA deletions in BCP-ALL patients. (A) Custom oligo array with high-density probe coverage at ch3q13.2, the RAG recognition sequences were identified within the deleted region. Each small vertical blue line indicates an exon. The thicker the line, the larger the exon. (B) Schematic representation of the CD200 and BTLA genes with locations of primers used for breakpoint-specific multiplex fluorescent PCR assay. Boxes represent genes, the arrows represent the primers and the star represents the fluorescent-labeled primer. Patients with mono-allelic deletion of both genes show both bands of F1/R and F2/R, patients with mono-allelic deletion of CD200 and bi-allelic deletion of BTLA show only the band of F1/R, patients with no deletion show only the band of F2/R. (C) Alignment of breakpoint sequences of five cases of CD200/BTLA deletion-positive BCP-ALL (n.1–5) and the REH cell line (n.6) with wild-type sequence showed that the genomic breakpoints of the CD200/BTLA deletions were conserved.

Figure 3.

Associations between CD200/BTLA deletions and outcome of all BCP-ALL patients treated according to the EORTC 58951 trial. Kaplan-Meier curves of (A) event-free survival, (B) disease-free survival and (C) overall survival in all BCP-ALL patients according to the presence of CD200/BTLA deletions.

Figure 4.

Associations between CD200/BTLA deletions and outcome in the intermediate-prognosis genetic risk group of BCP-ALL patients treated according to the EORTC 58951 trial. Kaplan-Meier curves of (A) event-free survival and (B) overall survival in the intermediate-prognosis genetic group according to the presence of CD200/BTLA deletions.