Mutations with loss of function of p53 are significantly associated with deletion or loss of 5q in t-MDS and t-AML after previous treatment with alkylating agents and are associated with genetic instability.

Two genes have been implicated in leukemias of patients with ab l of chromosome 3, band q26: EVII, which can be activated over long dances by chromosomal rearrangements involving 3q26, and EAP, a ribosomal gene that fuses withAMLI in a therapy-related myelodysplasia patient with a t(3;21)(q26.2;q22). AMLI was identified by its involvement in the t(8;21)(q22;q22) of acute myelod leukemia. Here we report the consistent identification of fusion transcripts between AMLI and EAP or between AMLI and previously unidentified sequences that we named MDSI (MDSassociated sequences) in the leukemic cells of four patients with therapy-related myelodysplala/acute myeloid leukemia and in one patient with chronic myelogenous leukemia in blast crisis, all of whom had a t(3;21). In addition, we have identified a third chimeric transcript, AMLI /EVI, in one ofthe therapyrelated acute myeloid leukemia patients.-field gel electrophoresis established the order of the genes as EAP, the most telomeric, and EVII, the most centromeric, gene. The results indicate that translocations could involve multiple genes and affect gene expression over long distances.The molecular analysis of recurring chromosomal translocations in leukemias has led to the identification of protooncogenes located at the translocation breakpoint that are activated either by altered expression or by gene fusion. One of the most common translocations in acute myeloid leukemia is the t(8;21)(q22;q22), which has recently been shown to involve the AMLI gene at 21q22 (1) fused to the ETO gene at 8q22 (2, 3). AMLI is identical' to the murine transcription factor pebp2a and the DNA-binding subunit of the human transcription core factor CBF (1, 4). The human and murine AMLi polypeptides are 99% homologous in their first 242 residues, but they differ in overall size. pebp2a encodes a predicted polypeptide of 452 residues containing the DNAbinding and dimerization region encoded by the Drosophila melanogaster runt (run) homology segment at the N terminus (2, 5), as well as a region rich in serine, threonine, and proline, suggestive of a transcription activation domain at the C terminus (4). The sequence ofthe human AMLI cDNA is 250 residues shorter than that of pebp2a and lacks the serine-, threonine-, and proline-rich region. It probably represents a spliced isoform of the mRNA consisting mostly of the run homology segment.Chromosome 21, band q22, is also involved in the t(3;21)(q26.2;q22) in therapy-related acute myeloid leukemia/myelodysplasia (t-AML/t-MDS) or chronic myelogenous leukemia in blast crisis (CML-BC) (6, 7). Recent studies have shown that this translocation involves the AMLJ gene and a gene at 3q26, which is EAP (8, 9). EAP codes for a small (129 amino acids) ribosomal protein, L22, and belongs to a large family of genes. Although EAP is highly conserved and abundantly expressed in all tissues (10, 11) and in hematopoietic cell lines (9), it is not known whether the allele at 3q26 is the one that is expressed. In t(3;21), the translocation results in th...

Myelodysplasia (MDS) and acute myeloid leukemia (AML) are heterogeneous, closely associated diseases arising de novo or following chemotherapy with alkylating agents, topoisomerase II inhibitors, or after radiotherapy. Whereas de novo MDS and AML are almost always subclassified according to cytogenetic characteristics, therapy-related MDS (t-MDS) and therapyrelated AML (t-AML) are often considered as separate entities and are not subdivided. Alternative genetic pathways were previously proposed in t-MDS and t-AML based on cytogenetic characteristics. An increasing number of gene mutations are now observed to cluster differently in these pathways with an identical pattern in de novo and in t-MDS and t-AML. An association is observed between activating mutations of genes in the tyrosine kinase RAS-BRAF signal-transduction pathway (Class I mutations) and inactivating mutations of genes encoding hematopoietic transcription factors (Class II mutations). Point mutations of AML1 and RAS seem to cooperate and predispose to progression from t-MDS to t-AML. Recently, critical genetic effects underlying 5qÀ/À5 and 7qÀ/À7 have been proposed. Their association and cooperation with point mutations of p53 and AML1, respectively, extend the scenario of cooperating genetic abnormalities in MDS and AML. As de novo and t-MDS and t-AML are biologically identical diseases, they ought to be subclassified and treated similarly.

Therapy-related acute myeloid leukemia (t-AML) in most cases develops after chemotherapy of other malignancies and shows characteristic chromosome aberrations. Two general types of t-AML have previously been identified. One type is observed after therapy with alkylating agents and characteristically presents as therapy-related myelodysplasia with deletions or loss of the long arms of chromosomes 5 and 7 or loss of the whole chromosomes. The other type is observed after therapy with topoisomerase II inhibitors and characteristically presents as overt t-AML with recurrent balanced chromosome aberrations. Recent research suggests that these 2 general types of t-AML can now be subdivided into at least 8 genetic pathways with a different etiology and different biologic characteristics. (Blood.

The p14 ARF , p15 INK4B , and p16 INK4A genes are important negative cell-cycle regulators often inactivated by deletions, mutations, or hypermethylation in malignancy. Hypermethylation of the three genes was studied in 81 patients with therapyrelated myelodysplasia (t-MDS) or acute myeloid leukemia (t-AML) by methylation-specific PCR, and p15 methylation additionally by bisulfite genomic sequencing. In all, 55 patients disclosed p15 methylation, five patients showed p16 methylation, whereas p14 methylation was not observed. Methylation of p15 was closely associated with deletion or loss of chromosome arm 7q (P ¼ 0.0006). In t-MDS, the p15 methylation frequency and the p15 methylation density both increased significantly by stage (P ¼ 0.004 and 0.0002), and p15 methylation frequency increased with an increasing percentage of myeloblasts in the bone marrow (P ¼ 0.006). In a two-variable Cox model including the percentage of myeloblasts, p15 methylation was an independent prognostic factor (P ¼ 0.005). Methylation of p15 was less common in t-AML of subtype M5 than in other FAB subtypes (P ¼ 0.03). Methylation of p15 was unrelated to type of previous therapy, to latent period from start of therapy, to platelet count, and to p53 mutations. Inactivation of p15 and deletion of genes on chromosome arm 7q possibly cooperate in leukemogenesis.

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The AML1 transcription factor is essential for normal hematopoiesis and is the target of several chromosomal translocations in acute leukemia. Acquired somatic AML1 mutations were recently demonstrated sporadically in de novo myelodysplasia (MDS) and acute myeloid leukemia (AML) including a few cases of therapy-related disease (t-MDS/t-AML). We examined 140 patients with t-MDS or t-AML for AML1 mutations by direct sequencing. We identified 9 missense, 3 nonsense, and 10 frameshift mutations, all heterozygous, in 22 patients (15.7%). Thirteen mutations were located in the N-terminal Runt homology domain (RHD), whereas 9 mutations were located in the C-terminal region including the transactivation domain (TAD). Nineteen patients with AML1 mutations had previously received alkylating agents whereas 2 patients had received radiotherapy only. AML1 mutations were highly significantly associated with presentation of the disease as t-MDS (P ‫؍‬ .003), with deletion or loss of chromosome arm 7q (P ‫؍‬ .001) and with subsequent transformation to overt t-AML (P ‫؍‬ .0001). Patients with missense mutations presented a shorter survival compared with patients with nonsense/frameshift mutations (P ‫؍‬ .03). Our results suggest that AML1 mutations and deletion of genes on chromosome arm 7q cooperate in leukemogenesis and predispose to leukemic transformation. (Blood.

Alternative genetic pathways were previously outlined in the pathogenesis of therapy-related myelodysplasia (t-MDS) and acute myeloid leukemia (t-AML) based on cytogenetic characteristics. Some of the chromosome aberrations, the recurrent balanced translocations or inversions, directly result in chimeric rearrangement of genes for hematopoietic transcription factors (class II mutations) which disturb cellular differentiation. Other genetic abnormalities in t-MDS and t-AML comprise activating point mutations or internal tandem duplications of genes involved in signal transduction as tyrosine kinase receptors or genes more downstream in the RAS-BRAF pathway (class I mutations). The alternative genetic pathways of t-MDS and t-AML can now be further characterized by a different clustering of six individual class I mutations and mutations of AML1 and p53 in the various pathways. In addition, there is a significant association between class I and class II mutations possibly indicating cooperation in leukemogenesis, and between mutations of AML1 and RAS related to subsequent progression from t-MDS to t-AML. Therapy-related and de novo myelodysplasia and acute myeloid leukemia seem to share genetic pathways, and surprisingly gene mutations were in general not more frequent in patients with t-MDS or t-AML as compared to similar cases of de novo MDS and AML studied previously.