Chromosome translocations and covert leukemic clones are generated during normal fetal development - PubMed (original) (raw)

Chromosome translocations and covert leukemic clones are generated during normal fetal development

Hiroshi Mori et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Studies on monozygotic twins with concordant leukemia and retrospective scrutiny of neonatal blood spots of patients with leukemia indicate that chromosomal translocations characteristic of pediatric leukemia often arise prenatally, probably as initiating events. The modest concordance rate for leukemia in identical twins ( approximately 5%), protracted latency, and transgenic modeling all suggest that additional postnatal exposure and/or genetic events are required for clinically overt leukemia development. This notion leads to the prediction that chromosome translocations, functional fusion genes, and preleukemic clones should be present in the blood of healthy newborns at a rate that is significantly greater than the cumulative risk of the corresponding leukemia. Using parallel reverse transcriptase-PCR and real-time PCR (Taqman) screening, we find that the common leukemia fusion genes, TEL-AML1 or AML1-ETO, are present in cord bloods at a frequency that is 100-fold greater than the risk of the corresponding leukemia. Single-cell analysis by cell enrichment and immunophenotype/fluorescence in situ hybridization multicolor staining confirmed the presence of translocations in restricted cell types corresponding to the B lymphoid or myeloid lineage of the leukemias that normally harbor these fusion genes. The frequency of positive cells (10(-4) to 10(-3)) indicates substantial clonal expansion of a progenitor population. These data have significant implications for the pathogenesis, natural history, and etiology of childhood leukemia.

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Figures

Figure 1

Schematic diagram of fusion transcripts and the relative location of amplification primers (arrows) for TEL-AML1 (A) and AML1-ETO (B).

Figure 2

Detection of TEL-AML1 fusion transcripts in normal cord blood samples by nested RT-PCR. (A) _TEL-AML1-_positive control cells (REH leukemic cell line) were serially diluted in negative cells (KG1 leukemic cell line). NTC was no template control. Sensitivity of detection of TEL-AML1 transcripts was 10−4. (B) Representative screening results (second-round RT-PCR) for TEL-AML1 fusion transcripts in cord blood. Two samples (B4, B243) were TEL-AML1 positive and six samples (B2, B3, B5, B242, B244, and B245) were negative. β2m, β2-microglobulin controls. (C) Representative fusion sequence of _TEL-AML1-_positive sample (B4). (D) RT-PCR analysis of different cell fractions from a _TEL-AML1-_positive cord blood (B4). β2-microglobulin (β2m) used as positive control. NTC was no template control.

Figure 3

Detection of AML1-ETO fusion transcripts in normal cord blood samples by nested RT-PCR. (A) _AML1-ETO-_positive control cells (Kasumi-1 leukemic cell line) were serially diluted in negative cells (HL-60 cell line). NTC was no template control. Sensitivity of detection of AML1-ETO transcripts was 10−5. (B) Representative screening results (second-round RT-PCR) for AML1-ETO fusion transcripts. One cord blood sample (B169) was positive and three others shown here (B167, B168, B170) were negative. NTC was no template control. β2m, β2-microglobulin controls. (C) Sequence of AML1-ETO derived from B169 cord blood cells.

Figure 4

Detection of TEL-AML1 and AML1-ETO fusion transcripts in normal cord blood samples by real-time quantitative PCR (Taqman). Shown is a linear detection of TEL-AML1 (A) and AML1-ETO (B) fusion transcripts (in REH and Kasumi-1 cell lines, respectively) over at least four and five logs, respectively. This assay could detect one _TEL-AML1-_positive cell in a background of 10,000 negative cells and one _AML1-ETO-_positive cell in a background of 100,000 negative cells. Cycle threshold values and positive cord bloods indicated with B or C numbers were used to calculate appropriate cell frequency (10−3 to 10−4) from standard.

Figure 5

Identification of TEL-AML1 fusion gene-positive cells in cord blood (B128) by immunophenotype/FISH analysis. In each case the fluorescent signal corresponding to the TEL probe is green, the AML1 probe is red, and the fused red-green signals corresponding to the TEL-AML1 fusion appear yellow. The cells positive for the corresponding immunophenotype are stained blue. (A) Positive control leukemic cell line REH, stained for CD10. Both CD10-positive cells show one fusion and two separate red signals. The smaller red signal corresponds to the translocated 5′ portion of the disrupted AML1 gene, and the larger red signal to the normal AML1 allele. Note that the green signal is absent because of deletion of the normal TEL allele in this cell line (as in most cases of ALL with TEL-AML1 fusion). (B) Negative control cell line Nalm 6, stained for CD10. Both CD10-positive cells show two green and two similar-size red signals, corresponding to two normal copies of TEL and AML1. (C) CD10-positive sorted cord blood (B128) cells. (Right) The cell shows one green, two different-sized red, and one (yellow) fusion signal, the expected signal configuration for a _TEL-AML1_-positive cell. The green (normal TEL) signal is present. (Left) The cell shows two green and two similar-sized red signals, corresponding to two normal copies of TEL and AML1. There is no fusion signal. (D) CD10−/CD34− cord blood cells stained for Ig κ. The κ-positive blue cell (Right) shows one yellow fusion, one green, and two different-sized red signals, the expected pattern for a TEL-AML1-positive cell. The normal TEL signal is present in this cell. The other cell (κ negative) shows two normal copies of TEL and AML1. (Magnifications: ×1,500.)

Figure 6

Identification of AML1-ETO fusion gene-positive cells in cord blood (B169) by immunophenotype FISH analysis. In each case the fluorescent signal corresponding to the AML1 probe is red and the ETO probe is green. The cells positive for the respective immunophenotypes are stained blue. (A) Positive t(8;21) cell line Kasumi. Both CD13/CD33-positive cells show two red-green (yellow) fusion signals, corresponding to the AML1-ETO and ETO-AML1 gene fusions, in addition to the single green and red signals of the normal AML1 and ETO alleles. (B) t(8;21) negative control: Epstein–Barr virus-transformed normal blood lymphocytes stained for CD19. Both cells show two normal copies each of AML1 and ETO. (C) Cord blood sorted mononuclear cells stained for CD13/CD33. The CD13/CD33-positive cell shown has one red, one green, and two yellow fusion signals, consistent with the presence of the t(8;21). (Magnifications: ×1,500.)

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Chromosome translocations and covert leukemic clones are generated during normal fetal development - PubMed (original) (raw)