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Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 1137-1138
CORRESPONDENCE
The Juxtaposition of ABL With BCR and Risk for
Fusion May Come at the Time of BCR Replication in Late
S-Phase
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LETTER |
To the Editor:
In their recent report in Blood, Neves et al1
demonstrated the close proximity of BCR and ABL in
hematopoietic cells in late S-phase of the cell cycle and provided a
basis for a model to explain the t(9;22) chromosomal abnormality. We
have studied the relative positioning of BCR and ABL in
cells that happened to be accumulating in late S- and
G2M-phase, and observed a similar proximity of the 2 genes.
In our evaluation, the juxtaposition appears to occur at the time of
the replication of the second of the 2 BCR loci.
The cells analyzed were from a 49-year-old man who had just begun
reinduction for a relapsed acute leukemia. The patient was initially
diagnosed morphologically as having acute myelogenous leukemia a year earlier at an outside hospital, but on transfer to the
University of Chicago was found to have circulating blasts (white blood
cell [WBC] count, 4.9 × 109/L, 8% blasts) with a
common precursor B-cell phenotype (CD19+,
CD10+, TdT+, CD34+,
sIg , CD13, and CD33 , MyPx).
Fluorescence in situ hybridization (FISH) analysis with probes for
MBCR and ABL (Oncor, Gaithersburg, MD) was performed on
a peripheral blood buffy coat smear (2% blasts) to determine if the
lymphoblasts were BCR/ABL+, and if so whether other
cell types were also positive.2 Inadvertently, the specimen
for FISH was obtained ~15 hours after the onset of therapy with
cyclophosphamide, daunorubicin, vincristine, and prednisone.
The FISH results, when correlated with the morphology of the previously
Wright-stained cells, showed no consistent BCR/ABL fusion
signals in the blasts or other cell types. This was supported by
cytogenetic and molecular analyses of an involved bone marrow, which
showed that the patient had a normal karyotype, and no BCR/ABL fusion transcripts for p190 or p210 by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The FISH results did show, however, that most of the blasts (16 of 27 evaluated) appeared to be in late S-
or G2M-phase, with many cells showing 3 or 4 copies
of both ABL and BCR (Fig 1A through
C). This cell-cycle distribution presumably
was an effect of the recently administered vincristine.3 The analysis also showed that the BCR and ABL signals
tended to approach one another when there were 6 or 7 total signals
(consistent with late S-phase) (Fig 1A), to merge at a time when the
second BCR seemed to be replicating (Fig 1B, and inset), and to
separate at G2M (Fig 1C). The distance between the 2 closest BCR and ABL signals was shortest (approaching 1 signal diameter length) when there were 6 or 7 total signals, and this
was statistically different from the shortest separation in cells with
4/5 or 8 total signals (P < .0001 and P < .001,
respectively).
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| Fig 1.
FISH analysis with a probe set for MBCR and
ABL, in blasts from a patient with recently treated acute
lymphoblastic leukemia shown to be Philadelphia-chromosome-negative
and BCR/ABL-negative. The digoxigenin-labeled probe for
BCR is detected with anti-dig rhodamine (red), and the
biotinylated probe to ABL is detected with fluoreceinated
avidin (green). The 3 illustrations represent cells in late S- (A and
B) and G2M-phase (C) of the cell cycle. In (A), there are 4 green and 3 red signals with close proximity of BCR/ABL
(inset). In (B), there are 4 green and 3 red signals, including a
doubling BCR (red) signal which is juxtaposed to ABL
(inset). The cell in (C) is at G2M and shows 4 green and 4 red signals with no juxtaposition of BCR and ABL.
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Our chance evaluation of cells from a patient who had already begun
therapy permitted us to observe BCR and ABL positioning in the otherwise infrequent cells of late S and G2M.
Although the small number of cells studied provide somewhat limited
data, our finding supports the results of Neves et al, showing close juxtaposition of the 2 genes in late S-phase of primitive hematopoietic cells. Our finding further advances their model by suggesting that the
actual juxtaposition of the 2 genes may occur around the time of the
replication of BCR. Our illustrations clearly show an approach,
convergence, and separation of the 2 genes when there is full
tetrapolid complement of ABL signals, and when BCR copy
number goes from 3 to 4. Although the close proximity of BCR
and ABL was identified in late S-phase cells in the work of Neves et al, the replication status or copy number of the BCR and ABL genes is not apparent from their data. In fact, it is curious that in their Fig 1, the late S-phase cell depicted seems to
have only a diploid complement (2 signals each) of BCR and ABL.
Our finding of BCR and ABL closest juxtaposition in
late S-phase at a time near an apparent BCR replication would
support the possibility of the BCR/ABL fusion developing during
a replication error in BCR. Recombination may more likely occur
during DNA replication, and this may be the case with regard to
the development of BCR/ABL and t(9;22).
John Anastasi
Rizwan Moinuddin
Christopher Daugherty
Departments of Pathology and Medicine
University of Chicago
Chicago, IL
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REFERENCES |
1.
Neves H, Ramos C, Gomes, da Silva M, Parreira A, Parreira L:
The nuclear topography of ABL, BCR, PML, RARa genes: Evidence for gene proximity in specific phases of the cell cycle and stages of hematopoietic differentiation.
Blood
93:1197, 1999[Abstract/Free Full Text]
2.
Anastasi J, Feng J, Dickstein JI, LeBeau MM, Rubin CM, Larson RA, Rowley JD, Vardiman JW:
Lineage involvement by BCR-ABL in Philadelphia chromosome positive lymphoblastic malignancies: Chronic myelogenous leukemia presenting in lymphoid blast phase versus Ph+ acute lymphoblastic leukemia.
Leukemia
10:795, 1996[Medline]
[Order article via Infotrieve]
3.
Hiller K, Meyer P, Wilms K:
In vivo cell kinetic effects of vincristine on the spontaneous AKR leukemia: recruitment of non-proliferating cells.
Blut
45:39, 1982[Medline]
[Order article via Infotrieve]
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