Expression of ERG is of prognostic significance in acute myeloid leukemia (AML) and T-lymphoblastic leukemia (T-ALL) pointing to its role in leukemogenesis. To unravel its transcriptional regulation we analyzed the expression of ERG specific isoforms. Expression of the two main isoforms ERG2 and ERG3 was found in AML and normal CD34+ cells, whereas T-ALL blasts only expressed ERG isoforms harboring exon 5 (ERG3) lacking expression of ERG2. Bisulfite sequencing revealed hypermethylation of a CpG island within the ERG2 promoter region in T-ALL. Treatment of the T-lymphoblastic cell line BE13 with decitabine led to re-expression of ERG2 and pyrosequencing showed concordant DNA hypomethylation, thus confirming a methylation regulated expression of ERG2. Moreover, the identification of a new ERG isoform (ERG3Deltaex12) suggests the association with different interaction partners and adds to the complexity of downstream pathways mediated by the expression of specific ERG transcripts in acute leukemia.

BackgroundNOTCH1 mutations have been associated with a favorable outcome in pediatric acute T-lymphoblastic leukemia However, the results of studies on the prognostic significance of NOTCH1 mutations in adult T-lymphoblastic leukemia remain controversial.Design and MethodsHere we have investigated the prognostic impact of mutations in the NOTCH1 pathway, in particular, the NOTCH1 and FBXW7 genes, in a large cohort of adult patients with T-lymphoblastic leukemia (n=126). We determined the occurrence Of Mutations In NOTCH1 and FBXW7 by DNA amplification and direct sequencing of polymerase chain reaction productsResultsMutations were identified in 57% and 12% of the NOTCH1 and FBXW7 genes, respectively. The characteristics of patients carrying NOTCH1 and/or FBXW7 (NOTCH1-FBXW7) mutations were similar to those with wild-type genes Patients with NOTCH1-FBXW7 mutations more often showed a thymic immunophenotype (p=0.001) In the over-all cohort; no significant differences were seen in the complete remission or event-free survival rates between patients with mutated or wild-type NOTCH1-FBXW7 (p=0.39)ConclusionsNOTCH1 and FBXW7 mutations were not predictive of outcome in the overall cohort of adult patients with T-lymphoblastic leukemia. but there was a trend towards a favorable prognostic impact of NOTCH1-FBXW7 mutations in the small subgroup of patients with low-risk ERG/BAALC expression status. Our findings further confirm the high frequency of NOTCH1 mutations in adult T-lymphoblastic leukemia

The oncogenic ETS transcription factor ERG is involved in various cellular pathways including developmental regulation, proliferation, and differentiation. In hematopoiesis ERG plays a specific role during normal T-cell differentiation showing high expression levels in stem cells and down regulation in the progenitor compartment. In this regard, it is intriguing that aberrant expression of ERG was found in a subset of patients with acute T-lymphoblastic leukemia (T-ALL) and was associated with an inferior outcome. Furthermore, high level ERG expression was of adverse prognostic significance in patients with newly diagnosed acute myeloid leukemia (AML), thus highlighting ERG's potential role in myeloid as well as T-lineage leukemogenesis. ERG3 (NM_182918) and ERG2 (NM_004449) represent the main isoforms and show abundant expression in myeloid and lymphoid hematopoietic progenitor cells. The expression pattern of specific ERG isoforms in acute leukemias has yet to be investigated. To further elucidate the nature of aberrant ERG expression we have determined the existence and transcriptional regulation of ERG isoforms in pretreatment bone marrow samples of adult T-ALL (n=21) and AML (n=20) patients as well as in normal CD34+ hematopoietic cells of healthy volunteers (n=5). 5′RACE revealed the presence of a new ERG isoform (ERG3Δex12) characterized by expression of exon 5 and absence of exon 12. Expression of ERG3Δex12 was verified by RT-PCR in AML, T-ALL, and CD34+ cells. In addition, real-time RT-PCR showed concomitant expression of the two main isoforms ERG2 and ERG3 in AML and normal CD34+ cells. In contrast, T-ALL patients lacked expression of ERG isoforms harboring exon 4 (ERG2). Promoter analyses of ERG2 and ERG3 revealed the presence of two CpG islands in the ERG2 promoter region, whereas no CpG island was predicted in the ERG3 promoter. Bisulfit conversion of genomic DNA and sequencing of cloned PCR products revealed a significantly higher degree of methylation of CpG island 2 in T-ALL samples (median: 86.4%, range: 16.0 – 98.8%) as compared to AML (median: 38.1%, range: 10.9 – 60.7%; P-value=0.0002 - two sided T-test). As for CpG island 1, CD34+ cells had the lowest rate of methylation in CpG island 2 (median: 7.7%, range: 2.4 – 20.7%). Thus, the differential expression of ERG isoforms is mediated by epigenetic silencing of exon 4 containing transcripts in T-ALL. In conclusion, the identification of the new ERG isoform (ERG3Δex12) suggests the association with different partners as the central exons, including exon 12, guide the interaction with different proteins. Furthermore, the distinct expression of specific ERG transcripts controlled by methylation adds to the complexity of ERG directed downstream pathways in different leukemic subtypes.

The NOTCH1 gene encodes a transmembrane receptor important for the T-lineage commitment. Activating NOTCH1 mutations had been found in approximately 56% of pediatric T-lymphoblastic leukemia (T-ALL) samples and were associated with a favourable outcome. Here the frequency and the association of NOTCH1 mutations to clinical features, as well as molecular markers were analyzed for the first time in a large cohort of adult T-ALL patients. Genomic DNA of pretreatment samples of 126 adult T-ALL patients enrolled on the GMALL protocols 05/93 and 06/99 were analyzed by direct sequencing for the presence of NOTCH1 mutations in the heterodimerization domain (HD-C, HD-N), the polypeptide enriched in proline, glutamate, serine and threonine (PEST; divided into PEST-1 and PEST-2) domain, and the transactivation domain (TAD). In addition, leukemic blasts were molecularly characterized for mRNA expression of HOX11, HOX11L2, ERG, and BAALC by real-time RT-PCR. Immunophenotyping was performed differentiating T-ALL into three subtypes (early, thymic, mature). NOTCH1 mutations were identified in 72 (57%) of the 126 patients including mutations in the HD (n=56), in the TAD (n=5), and in the PEST (n=20) domain. Eleven patients had more than one mutation, including 3 patients with HD and TAD, and 6 patients with coexisting HD and PEST mutations. T-ALL patients carrying mutations revealed similar clinical characteristics as compared to NOTCH1 wild type cases with respect to age, sex, CNS involvement, white blood count. Moreover, no differences were seen in the response to induction therapy or survival between NOTCH1 wild type and mutated cases (in the overall groups as well as in subgroups analyses). In the expression pattern of HOX11, HOX11L2, BAALC, or ERG there were no significant differences between the two NOTCH1 groups. Patients with mutated NOTCH1 showed a higher frequency of a thymic immunophenotype (65% vs. 39% for unmutated cases; P=0.013). Furthermore, a significant association was observed between the expression of specific surface antigens and the NOTCH1 genotype: NOTCH1 mutated cases were significantly associated with the expression of CD10 (P<0.001) and CD1a (P<0.001) as well as lack of CD117 (P=0.004) and CD34 expression (P=0.04). Interestingly, mutations in the HD-N domain were highly associated with the expression of CD10 (P<0.001), whereas mutations in the PEST-1 region were characterized by CD1a positivity (P<0.001). In contrast, no significant associations were seen for the HD-C, TAD, or the N-terminal PEST-2 domain to the surface marker expression. In conclusion, NOTCH1 mutations occur in all molecular defined subtypes (HOX11, HOX11L2, ERG, BAALC); thus these data suggest that NOTCH1 activation promotes transformation of T-cell progenitors as an early step independent of the additional, subsequent genetic alterations. Moreover, activating NOTCH1 mutations in adult T-ALL display a distinct leukemic phenotype suggesting that mutations in specific NOTCH1 domains arrest thymocytes at a distinct maturation stage. Recognition of this specific immunophenotype may direct the detection for NOTCH1 mutations and the guidance of treatment strategies as the high frequency of NOTCH1 mutations in adult T-ALL makes an ideal target for pharmacological interventions.