Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3634-3635
CORRESPONDENCE
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To the Editor: |
Detection of genetic markers on different populations of
hematopoietic progenitor cells in B-cell chronic lymphocytic
leukemia
B-cell chronic lymphocytic leukemia (B-CLL) results from the
clonal expansion of CD5+/CD23+ B-lymphocytes.
The malignant transformation is assumed to occur at the level of mature
B lymphocytes. Newer studies, however, have questioned this assumption.
Estrov et al were able to generate CLL B-lymphoid colonies in vitro
from CD34+ progenitor cells.1 Gahn et al have
detected genetic abnormalities, such as trisomy 12 and Rb1 deletions,
at the level of CD34+ progenitor cells,2 and
Faguet et al have found granulocytes and monocytes expressing common
CLL antigen in a large subset of B-CLL patients.3 In order
to further explore this important issue, we studied different cell
populations for the presence of certain genetic abnormalities, such as
deletions on chromosome 13q14, commonly found in B-CLL.4
Populations of progenitor cells were defined according to the
positivity to CD34 and AC133 antigens. AC133 is a novel marker that is
selectively expressed on a CD34bright subset of human
hematopoietic stem and progenitor cells including the noncommitted, the
majority of those committed to the granulocytic-monocytic pathway and
the long-term repopulating stem cells.5
Peripheral blood samples from 22 patients with B-CLL were tested. Ten
healthy individuals of the hospital staff served as controls. After
preparation of mononuclear cell suspensions, AC133+ and
CD34+ cells were positively selected by magnetic-activated
cell sorting (MACS) according to the manufacturer's directions. Three
different cell populations were thus separated by sequential selection: CD34+/AC133+, representing the more immature
progenitor cell fraction; CD34+/AC133
, the
less immature cell fraction; and
CD34/AC133, the fraction of more mature
cells. The purity of isolated progenitor cells was evaluated by
fluorescence microscopy, using CD34-FITC and AC133-PE monoclonal
antibodies, and was 90% to 98% in all instances. Each cell population
was examined for the presence of deletions of D13S25 (13q14.3), D13S319
(13q14.3), and Rb1 (13q14.2) genetic loci by combining fluorescence
immunophenotyping and dual color fluorescence in situ hybridization
(FISH). The study was performed on cytospin preparations, and signals
were enumerated in at least 300 interphase cells.
In the CD34/AC133 cell population,
heterozygous or homozygous deletions of D13S25 and D13S319 were found
in 10 out of 22 patients (45.5%) (Figure,
A) in a high proportion of cells (63%
to 95.8%). Rb1 deletions (all heterozygous) were observed in 6 out of
22 cases (27.3%) in 23% to 83% of cells. All 6 patients also
displayed deletions of D13S25 and D13S319. In the
CD34+/AC133 population, heterozygous
deletions of D13S25 and D13S319 loci were detected in 4 out of 22 patients (18.2%), in a relatively small proportion of cells (10%,
15%, 17%, and 18%, respectively) (Figure, B). Heterozygous Rb1
deletion was found in 1 patient, in 33% of cells. No cells with
deletions in any of the examined loci were detected in the
CD34+/AC133+ cell fraction (Figure, C). In
controls, hetero- (1 signal) and homozygous (no signal) deletions of
D13S25 and D13S319 could be detected in a mean ± 2 SD of only 1.5 ± 0.56 of cells and Rb1 deletions in a mean ± 2 SD of 0.5 ± 0.36 of cells.
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Detection of genetic markers by FISH in different cell populations
of patients with B-CLL.
(A) CD34/AC133 cells displaying (a)
heterozygous deletions of both Rb1 and D13S319, (b) heterozygous
deletion of Rb1 and homozygous of D13S319, and (c) heterozygous
deletion of only Rb1. Red dots (signals) correspond to the Rb1 gene and
green ones to the D13S319 locus. Yellow signals are produced by the
fusion of red and green signals from Rb1 and D13S319 lying close on the
same chromosome (13q14). (B) CD34+/AC133
cells (green surface staining) with different genetic profile: (a) with
presence of 2 red and 2 green signals (no deletions), (b) with
heterozygous deletion of D13S319, and (c) with heterozygous deletion of
both Rb1 and D13S319. (C) CD34+/AC133+ cells
(red surface staining) with no marker deletions (all 4 signals
present).
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In conclusion, we found in a subset of B-CLL patients genetic
abnormalities in a small but appreciable proportion of moderately immature progenitor cells (CD34+/AC133),
similar to those found in the more mature fraction of cells (CD34/AC133). Our findings support the
concept that malignant transformation in B-CLL may indeed involve a
population of early hematopoietic cells, though less immature than the
primitive progenitor cells. In accordance with that is our recent
finding that, in a large subset of B-CLL patients, dendritic cells
carry the same genetic markers as clonal lymphoid cells and are
probably neoplastic in origin.6 All these results are
significant for our understanding of B-CLL biology and for selection of
the proper fraction of progenitor cells for autologous transplantation
in this disease.
Anna Lazaridou
Chrysavgi Miraxtsi
Angelos Tokmaktsis
Evdoxia Hatjiharissi
John Christakis
Department of Hematology
"Theagenio" Cancer Center
Thesasloniki, Greece
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References |
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Identification of chronic lymphocytic leukemia (CLL) progenitor cells.
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Gahn B, Schafer C, Neef J, et al.
Detection of trisomy 12 and Rb-deletion in CD34+ cells of patients with B-cell chronic lymphocytic leukemia.
Blood.
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3.
Faguet GB, Mruthinti S, Moores R.
Flow cytometric evidence suggesting a pluripotent chronic lymphocytic leukemia (CLL) progenitor cell.
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4.
Liu Y, Hermanson M, Grander D, et al.
13q deletions in lymphoid malignancies.
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Yin AH, Miraglia S, Zanjani ED, et al.
AC133, a novel marker for human hematopoietic stem and progenitor cells.
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Lazaridou A, Miraxtsi Ch, Korantzis I, Christakis JI.
Dendritic cells in B-cell chronic lymphocytic leukemia are neoplastic and maybe of pluripotent stem cell origin.
Blood.
1999;94:118a.
