Establishment and Characterization of a tel/abl Rearrangement Responsible for Imatinib Sensitivity in bcr/abl Negative Acute Lymphoblastic Leukemia.

Proliferation of Philadelphia (Ph) positive acute lymphoblastic leukemia (ALL) cells is driven by the bcr/abl oncoprotein which renders transformed cells independent of exogeneous growth factors. Bcr/abl can be targeted by fusion tyrosine kinase inhibitors such as imatinib. ALL is a heterogenous disease. Some cases of Ph negative ALL may be driven by oncogenic fusion kinases other than bcr/abl, with or without involvement of the abl-gene. Such kinases may also be susceptible to targeted inhibitors. At present however, few of such fusion kinases are known. This may be due to the lack of reliable in vitro culturing systems for primary ALL cells. Because of this, only a limited number of ALL cell lines are available to screen for alternative oncogenic fusion kinases. We have recently established an in vitro culturing system in which continuous proliferation (>1 year) was thus far induced in 11 out of 22 primary ALL samples, in the absence of exogenously added growth factors. Five of these 11 cultures were Ph negative by cytogenetics and bcr/abl negative by RT-PCR. Therefore, we first screened these lines for sensitivity to imatinib. One of the 6 Ph negative cell lines, LeidenALL-VG, derived from a 30-year old patient with Ph chromosome negative common ALL, displayed sensitivity to imatinib with an IC50 of 0.1 μM, comparable to the sensitivity of the bcr/abl positive cell lines. RT-PCR with primers specific for tel and abl revealed tel/abl fusion transcripts. Sequencing of the amplicons showed two splice variants of tel-abl: tel4/abl2 and tel5/abl2. Multicolor FISH based karyotyping using COBRA-FISH did not provide evidence for the suggested t(9;12) translocation, indicating cryptic tel/abl rearrangement. To map cryptic genomic aberrations, an array-comparative genomic hybridization array (array-CGH) was performed. A region at 9q34 spanning two array clones was gained (clones RP11-83J21 and RP11-143H20), corresponding with gain of a 300-1300 kb region containing the distal part of the abl gene. A region at 12p13 spanning one array clone was lost (clone RP11-59H1) corresponding with loss of a 1150 to 1750 kb region directly centromeric to the tel gene. To localize cryptic tel/abl rearrangement, fluorescent in situ hybridization (FISH) was performed using the distal tel and abl probes identified by array-CGH, as well as the clones proximal to these regions. FISH revealed an additional distal abl signal, co-localized with the proximal tel signal, on the short arm of an otherwise normal looking chromosome 12. With this, tel/abl rearrangement in LeidenALL-VG cells was characterized as a micro-insertion of one additional distal abl region into the tel region of a normal chromosome 12. Tel/abl rearrangement and imatinib sensitivity were confirmed in the primary cells. These results suggest that our serum-free approach to in vitro culture of primary ALL cells, in combination with molecular karyotyping tools such as array-CGH and COBRA-FISH, may allow identification and characterization of otherwise un-identified chromosomal aberrations, such as the cryptic tel/abl translocation illustrated here, that may be targeted by kinase inhibitors such as imatinib.