AML1/ETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 8;21 chromosomal translocation - PubMed (original) (raw)

AML1/ETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 8;21 chromosomal translocation

T Miyamoto et al. Proc Natl Acad Sci U S A. 2000.

Abstract

Leukemia-specific AML1/ETO transcripts are detectable in most patients with t(8;21) acute myelogenous leukemia (AML) in long-term remission. To understand the inconsistency between the clinical cure and the presence of "residual disease" at a molecular level, we separated and identified the cells expressing AML1/ETO by phenotype and function. Here we demonstrate that AML1/ETO transcripts are present in a fraction of stem cells, monocytes, and B cells in remission marrow, and in a fraction of B cells in leukemic marrow, but not in T cells. AML1/ETO transcripts also were demonstrated in a fraction of colony-forming cells of erythroid, granulocyte-macrophage, and/or megakaryocyte lineages in both leukemic and remission marrow. These data strongly suggest that the acquisition of the t(8;21) occurs at the level of stem cells capable of differentiating into B cells as well as all myeloid lineages, and that a fraction of the AML1/ETO-expressing stem cells undergo additional oncogenic event(s) that ultimately leads to transformation into AML.

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Figures

Figure 1

Figure 1

HSC and CLP in remission and leukemic BM. Five-color flow cytometric analyses of BM cells in remission (A: case 5b) and in leukemic phase (B: case 5a). BM cells were first gated by the negative expression of lineage-related antigens. (A) The CD34+Thy-1+CD38-/lo HSC and CD34+Thy-1-CD10+CD38+ CLP were sorted from remission BM. (B) In leukemic BM, HSC and CLP were absent, whereas primitive CD34+CD38-/lo leukemic progenitors and CD34+CD38+ leukemic blasts existed. FSC, forward light scatter.

Figure 2

Figure 2

Detection of AML1/ETO+ cells in triple-sorted populations from remission or leukemic BM. (A) RT-PCR analysis on purified cells from remission BM. Five hundred HSC, CLP, monocytes, and B cells and 5,000 T cells were triple-sorted and subjected to RT-PCR analysis. Representative data in case 22, who had maintained remission for 80 months at the time of sampling, are shown. The AML1/ETO transcript was sometimes detectable in HSC, CLP, monocytes, and B cells, but not in T cells after the second round of PCR amplification; + and − under each lane depict positive and negative result of PCR, respectively. Data of all cases are summarized in Table 2. Note that MPO gene is not expressed in AML1/ETO+ pooled B cells and CLP, which confirms that the samples do not contain myelomonocytic cells. (B) RT-PCR analysis on purified cells from leukemic BM. Representative data in case 5a are shown. All 500 pooled CD34+CD38-/lo and CD34+CD38+ cells expressed AML1/ETO mRNA, which is detectable by the first round of PCR, and AML1/ETO+ B cells were found at a higher frequency compared with those in remission BM as summarized in Table 3. The last five right lanes show results of PCR analysis in two AML1/ETO+ and three AML1/ETO- EBV-transformed B cell lines established from case 1. Note that in these AML1/ETO+ B cell lines, AML1/ETO transcripts were detectable by the first round of PCR amplification. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 3

Figure 3

Frequency of AML1/ETO+ cells in B cells and monocytes estimated by limit dilution analyses. The percent of samples negative by RT-PCR for AML1/ETO transcripts is plotted on the y axis versus the number of cells per sample tested. According to the Poisson statistics, the frequency of AML1/ETO+ cells can be estimated as numbers of cells in samples that show 37% of detection failures (arrows). The frequency of AML1/ETO+ cells was estimated to be 1 in 41,000 (case 5) and 38,000 (case 6) mature B cells in remission marrow (○), and 1 in 5,100 (case 5) and 6,200 (case 6) mature B cells in leukemic marrow (●), indicating that leukemic BM contains t(8;21)+ B cells at ≈10-fold higher frequency, compared with remission BM. AML1/ETO+ cell was estimated to be in 1 in 3,800 (case 5) and 4,700 (case 6) monocytes in remission marrow (□).

Figure 4

Figure 4

AML1/ETO+ myeloid progenitors in the CD34+CD38-/lo fraction of leukemic BM. (A) Morphology of AML1/ETO+ myeloid colonies and AML1/ETO+ leukemic blast colonies derived from single AML1/ETO+ progenitors. These AML1/ETO+ colonies included colonies composed of CFU-L (a), CFU-GM (b), BFU-E (c), and CFU-Meg (d). (B) RT-PCR analysis of cells picked from single cell-derived colonies. a_–_d correspond to a_–_d in A. Note that erythrocyte and megakaryocyte colonies did not express MPO gene, which confirms that these colonies did not contain myelomonocytic components. The frequency of these AML1/ETO+ myeloid progenitors was up to 60% in total myeloid colonies as shown in Table 4. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 5

Figure 5

Gradual decrease in frequency of AML1/ETO+ progenitors along with remission duration. Twenty-one remission marrow samples from 19 patients who had maintained complete remission >5 years after the sampling were analyzed. Fifteen cases were treated with chemotherapy (●) and four cases were treated with autologous MBT (○). The frequency of AML1/ETO+ myeloid progenitors and remission duration at the time of sampling appeared to be inversely correlated by a Spearman rank correlation analysis (P = 0.0006, r = −0.907 in patients treated with chemotherapy; P = 0.2774, r = −0.486 in patients treated with autologous MBT; P = 0.0007, r = −0.751 in total patients). The correlation curve shows the result in patients treated with chemotherapy.

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