Array-CGH Analysis of T-ALL Patients and Cell Lines.

Background

Molecular analysis of T-cell acute lymphoblastic leukemia (T-ALL) has provided evidence that a stepwise alteration of at least four specific pathways is required during transformation of thymocytes to leukemic T-cells. Genetic alterations in hematopoietic precursor cells lead to loss of cell cycle control, impaired differentiation, proliferation and survival advantages, and unlimited self-renewal capacity. These defects include inactivation of CDKN2A (P16) present in 96 % of the patients, deregulated expression of transcription factors, and mutation of NOTCH1 in 56% of patients. However, the molecular lesions leading to the proliferative and survival advantages of T-ALL cells are less well characterized, remaining unknown in 80 % of the T-ALLs.

Aims

Our aim was to set up a genome-wide analysis of T-ALL in order to detect cryptic deletions and amplifications, with a special focus on the 90 protein tyrosine kinase genes present in the human genome.

Methods

We used the array-CGH (micro-array comparative genomic hybridization) technology with slides containing genomic BAC probes spaced every 1 Mb over the human genome. An additional 480 probes were added covering the genomic locations of each of the 90 protein tyrosine kinases genes. A total of 27 T-ALL cases and 12 cell lines were included in the study.

Results

An interstitial deletion on chromosome 9p24 directly upstream of JAK2 was identified in one patient. The deletion was confirmed by FISH. Quantitative PCR (qPCR) analyses indicated that the deletion was 700 kb in size including exons 1–4 of JAK2. In two cell lines, deletions affecting ALK and ERBB4 were detected. Molecular analyses to characterize the possible presence of fusion transcripts involving tyrosine kinases are in progress. We did not detected other rearrangements involving tyrosine kinase genes in neither of the 26 other T-ALL cases nor in the 10 cell lines, indicating that cryptic deletions or amplifications involving tyrosine kinase genes are relatively rare in T-ALL.

The most frequent aberration was the deletion of CDKN2A (16/27 cases and 9/12 cell lines) ranging from only one clone to almost the complete 9p arm. MYB duplication was found in two cases and 4 cell lines, and was confirmed by qPCR and FISH analysis. Two of the 4 cell lines had overexpression of MYB detected by qPCR, which can interfere with apoptosis enhancing the survival of the cells, as has been previously described. PTEN deletion was present in one case and one cell line. Other unbalanced aberrations of various size were detected: del (2p) in 5/12 cell lines, del (5q) in 1/27 samples and 4/12 cell lines, del(6q) in 3/27 samples and 2/12 cell lines, or del(9p) in 5/27 samples and 4/12 cell lines. Some of these rearrangements were not observed by standard cytogenetics. Strikingly, cell lines had significantly more chromosomal abnormalities than primary T-ALL cases.

Conclusions

We detected a novel cryptic rearrangement of JAK2 in one T-ALL case, and duplication of MYB in two T-ALL cases (2/27; 7.5%) and four cell lines (4/12; 33%). Our results suggest that cryptic deletions or amplifications involving tyrosine kinase genes are relatively rare in T-ALL.