Pathogenesis Of T-cell Acute Lymphoblastic Leukemia Research Articles

Fusion of EML1 to ABL1 in T-cell acute lymphoblastic leukemia with cryptic t(9;14)(q34;q32)

AbstractThe BCR-ABL1 fusion kinase is frequently associated with chronic myeloid leukemia and B-cell acute lymphoblastic leukemia but is rare in T-cell acute lymphoblastic leukemia (T-ALL). We recently identified NUP214-ABL1 as a variant ABL1 fusion gene in 6% of T-ALL patients. Here we describe the identification of another ABL1 fusion, EML1-ABL1, in a T-ALL patient with a cryptic t(9;14)(q34;q32) associated with deletion of CDKN2A (p16) and expression of TLX1 (HOX11). Echinoderm microtubule-associated protein-like 1-Abelson 1 (EML1-ABL1) is a constitutively phosphorylated tyrosine kinase that transforms Ba/F3 cells to growth factor-independent growth through activation of survival and proliferation pathways, including extracellular signal-related kinase 1/2 (Erk1/2), signal transducers and activators of transcription 5 (Stat5), and Lyn kinase. Deletion of the coiled-coil domain of EML1 abrogated the transforming properties of the fusion kinase. EML1-ABL1 and breakpoint cluster region (BCR)-ABL1 were equally sensitive to the tyrosine kinase inhibitor imatinib. These data further demonstrate the involvement of ABL1 fusions in the pathogenesis of T-ALL and identify EML1-ABL1 as a novel therapeutic target of imatinib. (Blood. 2005;105:4849-4852)

Amplification of the ABL Gene in T-Cell Acute Lymphoblastic Leukemia (T-ALL).

In acute leukemias gene amplification occurs with incidence of 1–10%. On conventional cytogenetic (CC) studies it is usually seen either intrachromosomally as homogeneously staining regions (HSRs) or extrachromosomally as double minute chromosomes (dmins). Fluorescence in Situ Hybridization (FISH) has established that the genes most frequently amplified in ALL are AML1 and MLL. Recently, the Leukaemia Research Fund UK Cancer Cytogenetic Group has detected the amplification of the ABL gene in 5/210 childhood and in 3/70 adult T-ALL and has suggested that this genetic abnormality might identify patients with a generally poor event-free survival (EFS). The present study was aimed at determining the incidence and clinical significance of ABL amplification in a series of 31 consecutive adult T-ALL patients. All of them had been submitted to routine FISH screening for BCR/ABL and TEL/AML1 fusions and for MLL amplification. ABL amplification was detected by chance in two patients (6.4%). In one CC did not yield analysable metaphases and in the other it showed the following karyotype:46,XX/46,XX,t(1;3)(p34;p21),del(6)(q21),del(7)(q32). FISH with a painting probe specific for chromosome 9 detected an occult trisomy in the patient without analysable mitosis and a normal pattern in the other one. In both patients the number of ABL signals varied from cell to cell and the observer was always unable to count them properly. FISH on mitotic cells showed that ABL additional copies were localized neither on chromosome 9 nor on any other chromosome and revealed that amplification was extrachromosomal in nature even if no dmins were visualized. Therefore, it was hypothesized that the amplified ABL sequences might be localized on submicroscopic extrachromosomal structures, the episomes. In order to check whether the ABL gene was really over-expressed we performed a quantitative RT PCR (Q RT PCR) assay using the β-2-microglobulin as reference gene and total RNA from a normal subject for calibration. Quantification was made using the DDCt method. By this way we found that on clinical diagnosis the two patients expressed the ABL gene nine and twelve times more than the control.From a clinical point of view both patients were males. They had a high white blood cell count (31.8 and 21.8x109/L); their blast cells exhibited a T-ALL immunophenotype and a L2 morphology; their lactic dehydrogenase level was elevated. One patient achieved a complete remission (CR) of fifteen month duration and relapsed while still on maintenance treatment, the other did not respond to chemotherapy. In the former patient ABL expression was normal in CR but increased again on disease recurrence. In conclusion our data show that i) FISH is absolutely required to identify a new subset of T-ALL patients characterized by ABL amplification, ii) the role of ABL amplification in T-ALL pathogenesis is still obscure, iii) large cooperative studies are required to better define the clinical outcome of these patients whose EFS seems to be poor, iiii) Q RT PCR might be used to quantify minimal residual disease.

Fusion of NUP214 to ABL1 on Amplified Extrachromosomal Elements in T-ALL.

The Philadelphia translocation, encoding the BCR-ABL1 (BCR-ABL) fusion gene, is typically found in chronic myeloid leukemia (CML) and precursor B-cell acute lymphoblastic leukemia (B-ALL), but is exceptionally rare in T-cell acute lymphoblastic leukemia (T-ALL). To study the potential involvement of ABL1 gene rearrangements in T-cell malignancies, we screened 90 T-ALL cases by fluorescence in situ hybridization (FISH), using BCR and ABL1 probes. No BCR-ABL1 fusion signals were observed, confirming the low frequency of this rearrangement in T-ALL, but we did observe marked amplification (> 10 signals per nucleus) ABL1 of in 5 of 90 (5.5 %) T-ALL patients.Amplification of ABL1 occurred on small extrachromosomal elements that were not detectable by conventional cytogenetics. and hence are referred to as episomes. FISH, and array-CGH analyses delineated the amplicon as a 500 kb region from chromosome band 9q34, containing the oncogenes ABL1 and NUP214 (CAN). Molecular analysis led to the identification of a NUP214-ABL1 fusion gene, which is generated as result of the circularization of the genomic region between ABL1 and NUP214 to form the episomes. This is the first example of an oncogenic fusion gene generated by extrachromosomal amplification. The NUP214-ABL1 transcript was detected in 5 patients with ABL1 amplification, in 5 of 85 (5.8 %) additional T-ALL patients, and in 3 of 22 T-ALL cell lines. The constitutively phosphorylated tyrosine kinase NUP214-ABL1 is sensitive to the tyrosine kinase inhibitor imatinib mesylate (STI-571). The recurrent cryptic NUP214-ABL1 rearrangement is associated with increased expression TLX1 of (HOX11) or TLX3 (HOX11L2), and with deletion of CDKN2A (p16), consistent with a multi-step pathogenesis of T-ALL. Our results identify a novel mechanism for the generation of a fusion gene on extrachromosomal elements, and indicate the importance of activated tyrosine kinase signaling in the pathogenesis of T-ALL. NUP214-ABL1 expression defines a new subgroup of T-ALL patients that could benefit from imatinib treatment.

Molecular Analysis of T-Cell Receptor Gene Rearrangements in Children with T-Cell Acute Lymphoblast Leukemia on DFCI ALL Consortium Protocol 95-01.

During normal T-cell differentiation, T-cell receptor (TCR) genes undergo recombination in an ordered way. TCRd genes rearrange first followed by TCRg genes, which may result in TCRgd+ T lymphocytes. TCRab T lymphocytes develop via a separate differentiation lineage, with TCRb preceding TCRa gene rearrangements. Unlike in normal T-cells, TCRd, TCRg and TCRb genes rearrangements can be detected in T-cell acute lymphoblastic leukemia (T-ALL) and these rearranged gene sequences can be used as molecular targets to detect minimal residual disease (MRD). Knowledge of the association between rearrangement patterns and disease pathogenesis of pediatric T-ALL is relatively limited. We analyzed 45 sequences of the TCR gene rearrangements (17 TCRd and 28 TCRg) obtained at presentation from 37 children with T-lineage ALL and these sequences were used as molecular targets to perform real-time PCR (RQ-PCR). Seven children had both TCRd and TCRg gene rearrangements. Sequence analysis revealed a higher frequency of Vd1-Jd1 rearrangements (88%) compared to Vd2-Jd1 (6%) and Vd2-Dd3 (6%). Of the 28 TCRg sequences, Vg2 gene segment was identified in 7 sequences, followed by Vg4 (5), Vg3 (4), Vg8 (4), Vg1 (2), Vg7 (2), Vg10 (2), Vg5 (1), and Vg9 (1). In keeping with previous findings the median number of nucleotides in the junctional regions was higher in TCRd (28bp) than in TCRg sequences (7.5bp, p<0.0001) suggesting random nucleotide insertions and that Dd3 genes contribute a longer junctional region in TCRd gene rearrangement. Detailed analysis of junctional region of TCRg sequences revealed that this region is larger in patients who also have a TCRd rearrangement (n=7, median 11bp) compared to those patients for whom only a TCRg sequences was found (n=21, median 5bp, p=0.02), perhaps explained by a recent finding that lack of expression of TdT was associated with the absence or shorter N sequences which contributed to shorter CDR3 regions in immature lymphocytes. Sequence specific junctional region primers were designed and amplified with J gene consensus primers and RQ-PCR performed using J regionconsensus probes. 13 of 14 TCRd targets (93%) reached a high reproducible sensitivity of 10−5 and 10−6, whereas 10 of 18 TCRg targets (56%) showed such sensitivity (p=0.044, Fisher's exact test). The junctional features of the TCR rearrangements for cases with only TCRg sequences and short N-regions were identified suggest that leukemic transformation is initiated at an early stage of T-cell ontology . The high junctional region diversity in TCRd rearrangements provides a better target to design patient specific primers (about 20bp) providing a higher sensitivity to detect MRD. RQ-PCR analysis was performed on 31 patients in whom BM (31) or PB samples (26) at day 30 were available for analysis. At this time point18 patients (58%) had no PCR detectable disease, six had MRD levels between 10−6 and 10−3 and 7 has MRD levels > 10−3. However, there was no difference in EFS in these three groups, and only one patient relapsed. We conclude that TCRg/d rearrangements provide suitable markers for RQ-PCR MRD analysis, but that in this treatment protocol high levels of MRD at the end of induction are not necessarily predictive of relapse in T-lineage ALL as they are in B-lineage ALL

Gain-of-Function NOTCH1 Mutations Occur Frequently in Human T Cell Acute Lymphoblastic Leukemia.

NOTCH1 was discovered originally through its involvement in a rare (7;9) translocation found in human T cell acute lymphoblastic leukemia (T-ALL). Here, we report that >50% of human T-ALLs have activating NOTCH1 mutations, occurring as amino acid substitutions in an extracellular heterodimerization (HD) domain and/or as frameshift and stop codon mutations that result in the deletion of a C-terminal PEST destruction box. Normal pro-NOTCH1 is processed into a heterodimer consisting of an extracellular subunit and a transmembrane subunit, which associate non-covalently through the HD domain. NOTCH1 activation is triggered by binding of Serrate or Delta-like ligands to the extracellular subunit, which induces successive proteolytic cleavages in the transmembrane subunit that are dependent on i) metalloproteases and ii) gamma-secretase. The γ-secretase cleavage releases intracellular NOTCH1 (ICN1), which translocates to the nucleus and forms a transcriptional activation complex with the transcription factor CSL and co-activators of the Mastermind family. Normal turnover of ICN1 is regulated by the C-terminal PEST sequence. Data pointing to the existence of frequent abnormalities of NOTCH1 in T-ALL stemmed from a functional screen of 30 T-ALL cell lines. This identified five T-ALL cell lines that underwent growth arrest in response to i) treatment with an inhibitor γ-secretase, and ii) retroviral transduction of dominant negative Mastermind-like-1. Sequencing of of cDNAs from 4 of these 5 cell lines demonstrated both a missense mutation in the HD domain and a frameshift mutation in the PEST domain lying in cis in the same NOTCH1 allele. Subsequent sequencing of genomic DNA obtained from bone marrow lymphoblasts of 96 children and adolescents with T-ALL demonstrated identical or similar mutations in NOTCH1 in 53 samples (55.2%). Mutations in the HD domain alone were observed in 26 cases (27.1%), in the PEST domain alone in 11 cases (11.4%), and in both the HD and PEST domains in 16 cases (16.7%). Mutations were observed in tumors associated with expression of HOX11 (2/3), HOX11L2 (10/13; 77%), TAL1 (12/31; 39%), LYL1 (9/14; 64%), MLL-ENL (1/3) or CALM-AF10 (1/2), which span the major molecular T-ALL subtypes. In contrast, NOTCH1 mutations were not observed in genomic DNAs samples obtained from B-ALL lymphoblasts (N=89), or from T-ALL patients with NOTCH1-associated disease at the time of clinical remission (N=4). Reporter gene assays conducted with plasmids expressing normal and mutated forms of NOTCH1 showed that a PEST deletion or various HD mutations alone caused ~1.5-fold and 3–9-fold stimulations of reporter gene activity, respectively, whereas normal NOTCH1 lacked intrinsic signaling activity. More strikingly, the combination of various HD mutations and a PEST deletion in cis caused synergistic 20–40-fold stimulations of reporter gene activity that were completely abrogated by a γ-secretase inhibitor, indicating that signaling depends on proteolysis. These results suggest a model in which HD domain mutations promote ICN1 production, and PEST domain mutations enhance ICN1 stability. Our findings greatly expand the role of NOTCH1 in the pathogenesis of human T-ALL, and provide a rationale for targeted therapies that interfere with NOTCH signaling.

Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia.

Very rare cases of human T cell acute lymphoblastic leukemia (T-ALL) harbor chromosomal translocations that involve NOTCH1, a gene encoding a transmembrane receptor that regulates normal T cell development. Here, we report that more than 50% of human T-ALLs, including tumors from all major molecular oncogenic subtypes, have activating mutations that involve the extracellular heterodimerization domain and/or the C-terminal PEST domain of NOTCH1. These findings greatly expand the role of activated NOTCH1 in the molecular pathogenesis of human T-ALL and provide a strong rationale for targeted therapies that interfere with NOTCH signaling.

Fusion of NUP214 to ABL1 on amplified episomes in T-cell acute lymphoblastic leukemia.

In T-cell acute lymphoblastic leukemia (T-ALL), transcription factors are known to be deregulated by chromosomal translocations, but mutations in protein tyrosine kinases have only rarely been identified. Here we describe the extrachromosomal (episomal) amplification of ABL1 in 5 of 90 (5.6%) individuals with T-ALL, an aberration that is not detectable by conventional cytogenetics. Molecular analyses delineated the amplicon as a 500-kb region from chromosome band 9q34, containing the oncogenes ABL1 and NUP214 (refs. 5,6). We identified a previously undescribed mechanism for activation of tyrosine kinases in cancer: the formation of episomes resulting in a fusion between NUP214 and ABL1. We detected the NUP214-ABL1 transcript in five individuals with the ABL1 amplification, in 5 of 85 (5.8%) additional individuals with T-ALL and in 3 of 22 T-ALL cell lines. The constitutively phosphorylated tyrosine kinase NUP214-ABL1 is sensitive to the tyrosine kinase inhibitor imatinib. The recurrent cryptic NUP214-ABL1 rearrangement is associated with increased HOX expression and deletion of CDKN2A, consistent with a multistep pathogenesis of T-ALL. NUP214-ABL1 expression defines a new subgroup of individuals with T-ALL who could benefit from treatment with imatinib.

Alterations of the p53, p21, p16, p15 and RAS genes in childhood T-cell acute lymphoblastic leukemia

We investigated the alterations of the p53, p21, p16, p15 and RAS genes in childhood T-cell acute lymphoblastic leukemia (T-ALL) and T-ALL cell lines by polymerase chain reaction–single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing. Mutations of the p53 gene were found in three of 57 (5%) patients at diagnosis, one of 14 (7%) patients at relapse and in 12 of 18 (67%) cell lines. In these 12 cell lines, four had more than two mutations of the p53 gene. The p53 mutations were found in four of five cell lines whose original fresh leukemic cells were simultaneously examined original fresh leukemic cells. However, only one of the four fresh leukemic cells had the same mutation. All patients with p53 mutations in the course of disease died. Mutations of the p21 gene were not identified in 71 fresh samples and in 18 cell lines. N-RAS mutations were found in two of 57 (4%) fresh T-ALL patients at diagnosis, and four of 18 cell lines (22%), whereas no mutations were detected in any samples at relapse. Alterations of the p16 gene were found in 18 of 47 (38%) patients at diagnosis and in seven of 14 (50%) at relapse. These differences were not statistically significant. There were no differences in the frequency of alteration of the p16 and p15 genes between event-free patients and the remaining patients. Furthermore, we found the methylation of p16 gene in three of seven patients lacking homozygous deletions, suggesting higher frequency of p16 inactivation than previous reports in T-ALL. Interestingly, we found that one allele is inactivated by methylation and another allele had nonsense mutation in one cell line (KOPT-K1), resulting in loss of protein expression of p16. This type of p16 inactivation has not been so far reported in leukemia. We conclude that, (1) p53 mutations are infrequent at diagnosis but tend to be associated with poor clinical outcome; (2) RAS and p21 mutations may not be involved in the pathogenesis of T-ALL; (3) not only frequent alterations of p16 and p15 genes but also methylation of p16 gene are involved in initiating the leukemogenesis of T-ALLs, and (4) these 5 genes are independently involved in T-ALL.

The role of chromosome translocations in T cell acute leukemia

A variety of unique chromosome translocations are found in the malignant cells of patients with T cell acute lymphoblastic leukemia (T-ALL). Molecular analysis of these translocations has implicated nine different proto-oncogenes in the pathogenesis of T-ALL. Despite the apparent genetic complexity of this disease, recent studies have uncovered a common pathway of T-ALL development that involves two distinct families of transcription factors.