Down syndrome acute lymphoblastic leukemia, a highly heterogeneous disease in which aberrant expression of CRLF2 is associated with mutated JAK2: a report from the International BFM Study Group

TLDR: The data suggest that the majority of DS children with ALL may benefit from therapy blocking the CRLF2/JAK2 pathways, and the gene expression signature of DS-ALL is enriched with DNA damage and BCL6 responsive genes, suggesting the possibility of B-cell lymphocytic genomic instability.

About: This article is published in Blood.The article was published on 2010-02-04 and is currently open access. It has received 304 citations till now. The article focuses on the topics: Immunoglobulin heavy locus & Regulation of gene expression.

Figures

Table 2. GSEA for genes of the DS-ALL expression signature, identified from the AIEOP dataset, in 3 other datasets

Table 3. Gene ontology pathways overrepresented in the differential DS-ALL signature

Figure 2. DS-ALL gene expression profile. (A) GSEA analysis on ICH dataset. Genes are ranked (bottom of panel, gray) according to their expression in DS-ALL samples versus the rest of the samples, by GSEA, using the default parameters. The members of a gene set S (here, the set of 535 genes down-regulated in DS-ALL, AIEOP data) are tested: are they randomly distributed in the ranked gene list, or primarily found at the top or bottom? Occurrences of members of the gene set S in the ranked gene list are shown as vertical black lines above the ranked signature. The green curve and upper y-axis represent the enrichment score (ES) as a function of the number of ranked genes tested for enrichment of gene set S. See supplemental Methods for full details. (B) Expression levels of the genes from the refined DS-ALL lists, measured on the AIEOP dataset. Four hundred twenty-three probe sets that belong to the refined DS-ALL profile gene lists are centered and normalized. Values for each individual case are represented by a color, with red representing deviation above the mean and blue representing deviation below the mean. The colors along the x-axis represent the different ALL subtypes, listed on the right of the plot. DS indicates Down syndrome ALL; HD, high hyperdiploid; TEL, TEL-AML1; BCR, BCR-ABL; E2A, E2A-PBX1; and MLL, MLL-AF4.

Figure 5. Functional significance of CRLF2 expression. (A) Cytokine withdrawal assay of BaF3 and BaF3-CRLF2 cells infected with either empty vector (EV), mouse FLAG-Jak2 wild-type (wt), or mouse FLAG-Jak2 R683S. Error bars represent SE. (B) Constitutive activation of the Jak/Stat5 pathway in BaF3 and BaF3-CRLF2 cells expressing mouse FLAG-Jak2 wild-type (wt) or R683S, after 5 hours of cytokine deprivation. IL3 indicates cells harvested after 5 hours of interleukin-3 deprivation followed by 15 minutes of interleukin-3 stimulation. (C) Effect of JAK inhibitor I on growth of BaF3 cells expressing Jak2 R683S and BaF3-CRLF2 cells expressing either wt Jak2 or Jak2 R683S.

Table 4. Enrichment of BCL6-related gene expression signatures and direct targets in DS-ALL profile genes (FDR < 15%)

Table 6. Clinical and diagnostic characteristics of patients with DS-ALL with high/medium expression of CRLF2 versus low/no expression of CRLF2

Frequently asked questions

Q1. What are the contributions in this paper?

The authors report gene expression and other analyses to elucidate the molecular characteristics of acute lymphoblastic leukemia ( ALL ) in children with Down syndrome ( DS ). Intriguingly, the gene expression signature of DS-ALL is enriched with DNA damage and BCL6 responsive genes, suggesting the possibility of B-cell lymphocytic genomic instability. Their data also suggest that the majority of DS children with ALL may benefit from therapy blocking the CRLF2/JAK2 pathways. 

Q2. What is the role of BCL6 in the DS-ALL expression profile?

BCL6 is a transcription factorexpressed primarily in mature B cells at the germinal centers, where it facilitates immunoglobulin (Ig) affinity maturation by repressing the DNA damage response. 

Q3. What was used as a template for the generation of CRLF2 mutations?

pMX-Puro-hCRLF2 was used as a template for the generation of CRLF2 mutations by site-directed mutagenesis (QuikChange-II-XL; Stratagene). 

Q4. What was the background of the CRLF2 staining?

For the calculation of delta mean fluorescence intensity (MFI), background nonspecific staining was evaluated in populations gated by CD19, comparing tubes with or withoutanti-CRLF2 antibodies. 

Q5. What did the authors do to identify the differential expression in the DS-ALL profile?

Pathway analysis and BCL6 signatureTo identify molecular pathways that showed differential expression in the refined DS-ALL profile, the authors interrogated the DAVID database23 of Gene Ontology functional categories. 

Q6. In what patients was CRLF2 expression analyzed in a diagnostic sample?

In 2 patients’ CRLF2 expression was analyzed in RNA derived from diagnostic and remission bone marrows and was seen only in the diagnostic sample. 

Q7. How did the authors refine their AIEOP-based lists?

The authors refined their AIEOP-based lists by including only genes that showed consistent expression patterns in at least 2 of the 3 other datasets. 

Q8. How many patients with DSALLs did the authors observe increased expression of CRLF2?

Confirming the expression of CRLF2 RNA and protein in DSALLs and extending these observations to patients for whom array data were not available, the authors observed increased expression of CRLF2 in 62% of 53 patients with DS-ALL. 

Q9. What is the mean fold change in DS-ALL?

One of the up-regulated genes is BCL6, with a mean fold change of 1.46 in DS-ALL compared with non DS-ALL (supplemental Table 4). 

Q10. What is the significance of CRLF2 in DS-ALL?

Russell et al13 reported aberrant expression of CRLF2 caused by either chromosomal translocations to the IGH@ locus or interstitial deletions upstream to CRLF2 juxtaposing CRLF2 with the P2RY8 regulatory elements in approximately 5% of childhood ALLs. 

Q11. What is the common method used for combining probe sets of the same gene?

For those genes that were represented by more than one probe set, the authors used, when needed, a combination procedure to create a single representation of a gene’s expression (supplemental Methods).