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Therapy-related acute myeloid leukaemia (t-AML) and therapy-related myelodysplastic syndrome (t-MDS) are well-recognized complications of cytotoxic chemotherapy and/or radiotherapy (1). There are several features that distinguish t-AML from denovo AML, including a higher incidence of TP53 mutations (2,3), abnormalities of chromosomes 5 or 7, complex cytogenetics and a reduced response to chemotherapy (4). However, it is not clear how prior exposure to cytotoxic therapy influences leukaemogenesis. In particular, the mechanism by which TP53 mutations are selectively enriched in t-AML/t-MDS is unknown. Here, by sequencing the genomes of 22 patients with t-AML, we show that the total number of somatic single-nucleotide variants and the percentage of chemotherapy-related transversions are similar in t-AML and denovo AML, indicating that previous chemotherapy does not induce genome-wide DNA damage. W e identified four cases of t-AML/ t-MDS in which the exact TP53 mutation found at diagnosis was also present at low frequencies (0.003-0.7%) in mobilized blood leukocytes or bone marrow 3-6 years before the development of t-AML/tMDS, including two cases in which the relevant TP53 mutation was detected before any chemotherapy. Moreover, functional TP53 mutations were identified in small populations of peripheral blood cells of healthy chemotherapy-naive elderly individuals. Finally, in mouse bone marrow chimaeras containing both wild-type and [Tp53.sup.+/-] haematopoietic stem/progenitor cells (HSPCs), the [Tp53.sup.+/-] HSPCs preferentially expanded after exposure to chemotherapy. These data suggest that cytotoxic therapy does not directly induce TP53 mutations. Rather, they support a model in which rare HSPCs carrying age-related TP53 mutations are resistant to chemotherapy and expand preferentially after treatment. The early acquisition of TP53 mutations in the founding HSPC clone probably contributes to the frequent cytogenetic abnormalities and poor responses to chemotherapy that are typical of patients with t-AML/t-MDS., t-AML and t-MDS are clonal haematopoietic disorders that typically develop 1-5 years after exposure to chemotherapy or radiotherapy (1). To understand better how prior cytotoxic therapy contributes to the high [...]

Most patients with acute myeloid leukaemia (AML) die from progressive disease after relapse, which is associated with clonal evolution at the cytogenetic level (1,2). To determine the mutational spectrum associated with relapse, we sequenced the primary tumour and relapse genomes from eight AML patients, and validated hundreds of somatic mutations using deep sequencing; this allowed us to define clonality and clonal evolution patterns precisely at relapse. In addition to discovering novel, recurrently mutated genes (for example, WAC, SMC3, DIS3, DDX41 and DAXX) in AML, we also found two major clonal evolution patterns during AML relapse: (1) the founding clone in the primary tumour gained mutations and evolved into the relapse clone, or (2) a subclone of the founding clone survived initial therapy, gained additional mutations and expanded at relapse. In all cases, chemotherapy failed to eradicate the founding clone. The comparison of relapse-specific versus primary tumour mutations in all eight cases revealed an increase in transversions, probably due to DNA damage caused by cytotoxic chemotherapy. These data demonstrate that AML relapse is associated with the addition of new mutations and clonal evolution, which is shaped, in part, by the chemotherapy that the patients receive to establish and maintain remissions., To investigate the genetic changes associated with AML relapse, and to determine whether clonal evolution contributes to relapse, we performed whole-genome sequencing of primary tumour-relapse pairs and matched skin samples [...]

A study was conducted to evaluate whether DNMT3A is recurrently mutated in acute myeloid leukemia samples and whether DNMT3A mutations are associated with poor survival rates. Results indicated that sNMT3A mutations are highly recurrent in such patients and are independently associated with poor outcomes.

Cytogenetic analysis of acute myeloid leukemia (AML) cells has accelerated the identification of genes important for AML pathogenesis. To complement cytogenetic studies and to identify genes altered in AML genomes, we performed genome-wide copy number analysis with paired normal and tumor DNA obtained from 86 adult patients with de novo AML using 1.85 million feature SNP arrays. Acquired copy number alterations (CNAs) were confirmed using an ultra-dense array comparative genomic hybridization platform. A total of 201 somatic CNAs were found in the 86 AML genomes (mean, 2.34 CNAs per genome), with French-American-British system M6 and M7 genomes containing the most changes (10-29 CNAs per genome). Twenty-four percent of AML patients with normal cytogenetics had CNA, whereas 40% of patients with an abnormal karyotype had additional CNA detected by SNP array, and several CNA regions were recurrent. The mRNA expression levels of 57 genes were significantly altered in 27 of 50 recurrent CNA regions AML | array CGH | genomics | SNP array

Acute myeloid leukaemia is a highly malignant haematopoietic tumour that affects about 13,000 adults in the United States each year. The treatment of this disease has changed little in the [...]

We previously developed a murine model of acute promyelocytic leukemia (APL) by using human cathepsin G gene regulatory elements to direct the expression of promyelocytic leukemia (PML)/retinoic acid receptor [alpha] (RAR[alpha]) and RAR[alpha]/PML fusion cDNAs to the early myeloid compartment of transgenic mice. To study the efficacy of noncytotoxic therapy in this animal model, cohorts of naive immunocompetent mice were inoculated with primary murine APL cells from a frozen tumor bank. Arsenic trioxide and liposomally encapsulated all-trans-retinoic acid (Lipo ATRA), alone or in combination, were administered for 21 days by i.p. injection using doses that yielded plasma levels similar to those observed in human APL patients treated with these agents. Lipo ATRA was highly effective in inducing durable molecular remissions in immunocompetent mice [C57BL/6 x C3H [F.sub.1] (B6C3HF1)]; arsenic therapy was much less effective, and did not clearly synergize with Lipo ATRA to increase the remission rate in immunocompetent mice. The survival of Lipo ATRA-treated severe combined immunodeficient (SCID) animals (lacking functional T and B cells) was inferior to that of immunocompetent B6C3HF1 recipients (40% vs. 88% survival at 1 y, P < 0.001). These data suggest that adaptive immunity cooperates with pharmacologic therapy to induce or maintain remissions in murine APL. It also implies that immunosuppressive anti-leukemia therapies could paradoxically blunt effective anti-leukemia immune responses that are important for clearing small numbers of residual tumor cells after chemotherapy-mediated cytoreduction.

We previously generated a transgenic mouse model for acute promyelocytic leukemia (APL) by expressing the promyelocytic leukemia (PML)-retinoic acid receptor (RAR[Alpha]) cDNA in early myeloid cells. This fusion protein causes a myeloproliferative disease in 100% of animals, but only 15-20% of the animals develop acute leukemia after a long latency period (6-13 months). PML-RAR[Alpha] is therefore necessary, but not sufficient, for APL development. The coexpression of a reciprocal form of the fusion, RAR[alpha]-PML, increased the likelihood of APL development (55-60%), but did not shorten latency. Together, these results suggested that additional genetic events are required for the development of APL. We therefore evaluated the splenic tumor cells from 18 transgenic mice with APL for evidence of secondary genetic events, by using spectral karyotyping analysis. Interstitial or terminal deletions of the distal region of one copy of chromosome 2 [del(2)] were found in 1/5 tumors expressing PML-RAR[Alpha], but in 11/13 tumors expressing both PML-RAR[Alpha] and RAR[Alpha]-PML (P [is less than] 0.05). Leukemic cells that contained a deletion on chromosome 2 often contained additional chromosomal gains (especially of 15), chromosomal losses (especially of 11 or X/Y), or were tetraploid (P [is less than or equal to] 0.001). These changes did not commonly occur in nontransgenic littermates, nor in aged transgenic mice that did not develop APL. These results suggest that expression of RAR[alpha]-PML increases the likelihood of chromosome 2 deletions in APL cells. Deletion 2 appears to predispose APL cells to further chromosomal instability, which may lead to the acquisition of additional changes that provide an advantage to the transformed cells.

Acute promyelocytic leukemia (APML) most often is associated with the balanced reciprocal translocation t(15;17) (q22;q11.2) and the expression of both the PML-RAR[Alpha] and RAR[Alpha]-PML fusion cDNAs that are formed by this translocation. In this report, we investigated the biological role of a bcr-3 isoform of RAR[Alpha]-PML for the development of APML in a transgenic mouse model. Expression of RAR[Alpha]-PML alone in the early myeloid cells of transgenic mice did not alter myeloid development or cause APML, but its expression significantly increased the penetrance of APML in mice expressing a bcr-1 isoform of PML-RAR[Alpha] (15% of animals developed APML with PML-RAR[Alpha] alone vs. 57% with both transgenes, P [is less than] 0.001). The latency of APML development was not altered substantially by the expression of RAR[Alpha]-PML, suggesting that it does not behave as a classical 'second hit' for development of the disease. Leukemias that arose from doubly transgenic mice were less mature than those from PML-RAR[Alpha] transgenic mice, but they both responded to all-trans retinoic acid in vitro. These findings suggest that PML-RAR[Alpha] drives the development of APML and defines its basic phenotype, whereas RAR[Alpha]-PML potentiates this phenotype via mechanisms that are not yet understood.