Combined interphase fluorescence in situ hybridization elucidates the genetic heterogeneity of T-cell acute lymphoblastic leukemia in adults

Abstract

Background: Molecular lesions in T-cell acute lymphoblastic leukemias affect regulators of cell cycle, proliferation, differentiation, survival and apoptosis in multi-step pathogenic pathways. Full genetic characterization is needed to identify events concurring in the development of these leukemias.

Design and methods: We designed a combined interphase fluorescence in situ hybridization strategy to study 25 oncogenes/tumor suppressor genes in T-cell acute lymphoblastic leukemias and applied it in 23 adult patients for whom immunophenotyping, karyotyping, molecular studies, and gene expression profiling data were available. The results were confirmed and integrated with those of multiplex-polymerase chain reaction analysis and gene expression profiling in another 129 adults with T-cell acute lymphoblastic leukemias.

Results: The combined hybridization was abnormal in 21/23 patients (91%), and revealed multiple genomic changes in 13 (56%). It found abnormalities known to be associated with T-cell acute lymphoblastic leukemias, i.e. CDKN2A-B/9p21 and GRIK2/6q16 deletions, TCR and TLX3 rearrangements, SIL-TAL1, CALM-AF10, MLL-translocations, del(17)(q12)/NF1 and other cryptic genomic imbalances, i.e. 9q34, 11p, 12p, and 17q11 duplication, del(5)(q35), del(7)(q34), del(9)(q34), del(12)(p13), and del(14)(q11). It revealed new cytogenetic mechanisms for TCRB-driven oncogene activation and C-MYB duplication. In two cases with cryptic del(9)(q34), fluorescence in situ hybridization and reverse transcriptase polymerase chain reaction detected the TAF_INUP214 fusion and gene expression profiling identified a signature characterized by HOXA and NUP214 upregulation and TAF_I, FNBP1, C9orf78, and USP20 down-regulation. Multiplex-polymerase chain reaction analysis and gene expression profiling of 129 further cases found five additional cases of TAF_I-NUP214-positive T-cell acute lymphoblastic leukemia.

Conclusions: Our combined interphase fluorescence in situ hybridization strategy greatly improved the detection of genetic abnormalities in adult T-cell acute lymphoblastic leukemias. It identified new tumor suppressor genes/oncogenes involved in leukemogenesis and highlighted concurrent involvement of genes. The estimated incidence of TAF_I-NUP214, a new recurrent fusion in adult T-cell acute lymphoblastic leukemias, was 4.6% (7/152).

Figure 1.

Representative FISH detection of A) TLX3 unbalanced translocation in patient n. 21: the TLX3 assay gives one fusion and one orange signal. (B) Trisomy of 6q including C-MYB in patient n. 6: clone RP1-32B1 spanning C-MYB gives three orange signals. The green signal corresponds to RP1-258B3 for GRIK2 which, in this case, underwent monoallelic deletion. (C and D) Unbalanced der(7)t(7;10)/TCRB-HOX11 in patient n. 14: the TCRB assay gives 1 fusion and 1 orange signal (C) and the double-color assay combining RP11-1220K2 (green) and RP11-107I14 (orange) results in one fusion, two orange and one green signal (D). Panels E and F) Cryptic del(9)(q34) in patient n. 3: metaphase FISH with the LSI BCR-ABL1 produces two green and one orange signals (E); a double-color experiment with RP11-544A12 orange and RP11-143H20 green shows one fusion signal on normal 9 and an orange signal on del(9)(q34) (F).

Figure 2.

Characterizing TAF_I-NUP214 fusion transcripts. Panel A) TAF_I-NUP214 fusion transcript cloning and sequencing in patients 3 (upper schema) and 4 (lower schema). Arrows indicate the primers used in PCR amplification. Panel B) Multiplex PCR screening study used specific primers for E2A control gene amplification (680 bp). RA multiplex PCR: Upper gel includes oligonucleotides listed in right boxes. TAF_I-NUP214 fusion transcripts were confirmed in patients 3 (280 bp) and 4 (121 bp) and detected in another three patients, i.e. X (121 bp) and Y (121 bp) and W (121bp). RB multiplex PCR: Lower gel includes oligonucleotides listed in right boxes. Only the control gene was amplified. Panel C) TAF_I nucleotide 353 (exon Iα) is fused in-frame to TAF_I nucleotide 77 (exon 2) in the TAF_Iα/NUP214 isoform. TAF_I nucleotide 76 (exon Iβ) is fused in-frame to TAF_I nucleotide 77 (exon 2) in the TAF_Iβ-NUP214 isoform. Sequence numbers refer to GenBank accession NM_003011.1 for TAF_I and NM_005085.2 for NUP214. The TAF_I exon I_ sequence is reported as NM_001122821.1.

Figure 3.

Gene expression profiling A) GEP of 24 adult T-ALL samples. Samples bearing TAF_I-NUP214 are characterized by a specific signature. Each row represents a probe set, each column represents a single sample. The color scale indicates the relative levels of expression: dark blue the lowest levels of expressions, red the highest levels of expression. Red indicates high expression and blue low expression. B) Expression of HOXA cluster genes resulted from ANOVA (P<0.005). Panel C) Expression levels of genes differentially expressed between TAF_I-NUP214 and T-ALL samples.