Expression of AML1/MTG8 transcripts in clonogenic cells grown from bone marrow of patients in remission of acute myeloid leukaemia with t(8;21)

Abstract

Summary.Patients in long-term remission of acute myeloid leukaemia (AML) M2 with t(8;21) after chemotherapy, with or without bone marrow transplantation, are known to retain residual cells which express AML1/MTG8 transcripts in bone marrow, detectable by RT-PCR. In order to determine whether these residual cells are clonogenic, we have grown remission bone marrow samples in standard semi-solid culture and picked individual CFU-GM and BFU-E colonies which were then analysed for the expression of AML1/MTG8 transcripts using a rapid specific RT-PCR technique. Nine patients were tested in remission, six between 1 and 83 months post chemotherapy, one 103 months post autologous bone marrow transplant and one 41 months post allogeneic bone marrow transplant. One of these patients also had quantitation of AML1/MTG8 transcripts on five occasions after recovery from each course of chemotherapy and at the end of treatment. There was a significant correlation between the percentage of positive colonies and the level of AML1/MTG8 transcripts. Between two and 80 CFU-GM and between two and 21 BFU-E colonies were analysed from each patient sample; 0-23% CFU-GM and 0-17% BFU-E colonies were found to express AML1/MTG8 transcripts suggesting that these residual cells are clonogenic in vitro and that the cell of origin is a multipotent myeloid progenitor.Keywords: acute myeloid leukaemia, t(8;21), RT-PCR, colonies.The FAB M2 subtype of acute myeloid leukaemia (AML) is closely associated with the translocation t(8;21)(q22;q22) involving the AML1 gene on chromosome 21 and the MTG8 (ETO) gene on chromosome 8, producing the fusion gene AML1/MTG8 (AML1/ETO) (Erikson et al, 1992). Transcripts of this fusion gene can be specifically and sensitively detected by the reverse transcriptase polymerase chain reaction (RT-PCR) (Downing et al, 1993;Nucifora et al, 1993a). It is well established that residual cells expressing AML1/MTG8 transcripts can be detected by RT-PCR in the bone marrow of patients in long-term remission of AML M2 with t(8;21) after chemotherapy alone and after autologous and allogeneic bone marrow transplantation (BMT) (Nucifora et al, 1993b;Kusec et al, 1994;Saunders et al, 1994). This chromosomal translocation is associated with good response to chemotherapy and long-term survival with which the presence of these cells is obviously compatible.However, it is not fully established whether these residual cells bearing the t(8;21) are part of the previous leukaemic clone, or whether they have clonogenic potential or belong to a non-myeloid lineage. It is also of interest to ascertain whether transcripts are first expressed in a cell committed to the granulocyte/macrophage lineage or in a multipotent progenitor.In this study we have analysed bone marrow (BM) from nine patients in remission of AML M2 who were positive for t(8;21) at presentation. We tested samples for the presence of t(8;21) by qualitative RT-PCR and a number of samples by quantitative RT-PCR. We have also grown bone marrow mononuclear cells in methylcellulose c...

Acute myeloid leukemia stem cells and CD33-targeted immunotherapy

“…18 Thus, preleukemic stem cells and LSCs may coexist and vary in their differentiative and proliferative properties and perhaps their response to therapy, that is, persistence of preleukemic stem cells but eradication of fully transformed LSCs. This could explain why AML1-ETO transcripts remain detectable in the blood and, at a low frequency, in various clonogenic progenitors in many patients with AML1-ETO AML in long-term remission, [55][56][57][58] whereas a second mutation (eg, KIT) becomes undetectable despite similar detection sensitivities. 67 The persistence of preleukemic stem cells is similarly supported by X chromosome inactivation studies showing persistently clonal hematopoiesis in some patients in morphologic remission after treatment for AML.…”

“…Also consistent with the notion of a founder mutation, or at least a very early event, is the demonstration of AML1-ETO in other patients in long-term remission after therapy for CBF AML. [55][56][57][58] Furthermore, the observation that aberrant CEBPA, DNMT3A, IDH1/2, and possibly NPM1 alleles are typically present at diagnosis and relapse suggests that these could be very early, possibly preleukemic, mutational events. [59][60][61][62] In contrast, the mutation status of FLT3, NRAS/KRAS, and WT1 often changes between diagnosis and relapse, and mutations may only be present in a subset of leukemic blasts, indicating a role as secondary events.…”

The Aml1‐eto Chimaeric Transcription Factor in Acute Myeloid Leukaemia: Biology and Clinical Significance

“…Several studies have demonstrated persistent expression of the AML1-ETO chimaeric mRNA in patients in long-term remission, including those treated with allogeneic bone marrow transplantation (Nucifora et al, 1993d;Guerrasio et al, 1995;Saunders et al, 1996;Jurlander et al, 1996;Miyamoto et al, 1996). In addition, persistent expression of AML1-ETO was detected in the clonogenic haemopoietic progenitors isolated from remission bone marrow samples obtained from patients who had been in remission off therapy for several years (Miyamoto et al, 1996;Saunders et al, 1997). Importantly, AML1-ETO expression was detected not only in myeloid/monocytic colonies, but also in erythroid colonies, suggesting that despite being in remission the bone marrow of these patients had retained viable multipotential haemopoietic progenitors containing t(8;21).…”

AML1 and the AML1-ETO fusion protein in the pathogenesis of t(8;21) AML

“…(3) AML1-ETO can be detected in the peripheral blood and marrow of patients with long-term complete remissions after treatment with chemotherapy or stem cell transplantation (Kusec et al, 1994;Miyamoto et al, 1995;Nucifora et al, 1993b;Saunders et al, 1994Saunders et al, , 1996. AML1-ETO positive, multipotent progenitor cells that yield normal blood cell colonies, including B cells can be demonstrated in these patients along with normal progenitors (Guerrasio et al, 1995;Miyamoto et al, 1996Miyamoto et al, , 2000Saunders et al, 1997). This strongly suggests that the AML1-ETO fusion event occurs in an early stem cell or progenitor cell and that additional events are required for the cell to become fully transformed.…”