Implication of HOXA9, MEIS1, AML1 and IRF4 In Childhood Acute Leukemia (AL) In Greek Patients

Abstract

AbstractAbstract 4643 Introduction:Childhood AL is characterized by abnormal chromosomal translocations and gene fusions. The most common cytogenetic aberrations are: TEL/AML1 (25%), E2A/PBX1 (4.8%), BCR/ABL (1.6%), MLL rearrangements (1.6%) and AML1/ETO (exclusively found in acute myeloid leukemia (AML)). Their high prognostic significance determines the intensification (eg. for MLL rearrangements) or de-intensification (eg. for TEL/AML1) of therapy, even though the mechanisms through which these fusions affect the prognostic outcome are largely unknown. However, the majority (~70%) of childhood patients is negative for these translocations and, even though most present with numeric cytogenetic abnormalities (eg. aneuploidies) at diagnosis, they are treated with the same therapeutic protocols as those with known translocations. Only BCR/ABL+ AL patients receive individualized treatment (addition of imatinib mesylate) that offers a better prognostic outcome, thus representing an example of how childhood leukemia can benefit from individualized therapy. The study of the genes implicated in leukemic lesions can give insight into the pathogenesis of leukemia and hence provide new diagnostic markers and therapeutic targets. It is therefore fundamental to make scientific hypotheses based on the behaviour of such genes and then perform experiments to validate or reject them. In the present study we investigate the expression of 4 such genes: HOXA9 and MEIS1, implicated in both normal hematopoiesis and MLL+ leukemias; AML1, an important regulator of B-cell differentiation and the most frequent target of chromosomal translocations; and IRF4, a transcription factor of lymphocyte differentiation, also implicated in leukemias and lymphomas. The aim of this study is to examine whether there is a correlation between the expression of the above genes and the leukemic cytogenetic profile. Materials and Methods:RNA was extracted with Trizol (Invitrogen Inc.) from bone marrow samples of 43 newly diagnosed children with leukemia (39 acute lymphoblastic leukemia (ALL) and 4 AML), 20 healthy children (controls) and from 4 cell lines, each representing a different type of childhood leukemia: CCRF-CEM (T-cell ALL), CCRF-SB (B-cell ALL), Reh (non-T, non-B ALL) and THP-1 (acute monocytic leukemia). Gene expression was investigated with Real-Time Reverse Transcription PCR (qRT-PCR), using the Plexor one-step qRT-PCR kit (Promega Inc.). Fluorescence in situ hybridization (FISH) was performed with dual-color probes (Abbott Molecular, Inc.). Results:Cytogenetic data were available for 41 patients (pts): TEL/AML1+: 5 pts (ALL) (12.2%), MLL+: 4 pts (3 ALL and 1 AML) (9.7%), BCR/ABL+: 3 pts (ALL) (7.3%), E2A/PBX+: 2 pts (ALL) (4.8%), AML1/ETO+: 1 pt (AML); the remaining 26 patients (63%) were negative for these fusions but 6 (14.6%) had extra copies of the AML1 gene. Gene expression was as follows: HOXA9: up-regulated in 20 patients (46.51%) and the CCRF-SB, Reh and THP-1 cell lines; MEIS1: up-regulated in 19 patients (44.19%); AML1: up-regulated in 21 patients (48.84%) and all 4 cell lines; IRF4: up-regulated in 25 patients (58.14%) and down-regulated in the CCRF-SB cell line. Simultaneous co-expression of HOXA9 and MEIS1 was present in 15 patients (34.88%). Of the 6 patients having extra copies of AML1, only one showed over-expression of the gene. Conclusions:In our group of patients the frequency of the TEL/AML1 fusion appears to be less, whereas the frequency of the MLL rearrangement appears to be higher, than that reported for western European countries. We find all of the 4 genes studied significantly up-regulated in certain groups of patients, as compared to controls, regardless of leukemic subtype. Up-regulation of HOXA9 and MEIS1 did not appear to be an exclusive characteristic of the MLL+ group. In the case of the AML1 gene, we find that gene amplification does not correlate with over-expression of the gene. In addition, IRF4, a known tumor suppressor gene which would be expected to be down-regulated in newly diagnosed leukemias, seems to be the one most frequently up-regulated (58.14% of patients). Our findings lead us to suggest that there might be common patterns of aberrant gene expression among childhood leukemia patients regardless of cytogenetic subtype. Such patterns could be further investigated in an attempt to gain insight into the etiology of leukemogenesis. Disclosures:No relevant conflicts of interest to declare.