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Prepublished online as a Blood First Edition Paper on October 24, 2002; DOI 10.1182/blood-2002-07-2341.
 Previous Article | Table of Contents | Next Article 
Blood, 1 March 2003, Vol. 101, No. 5, pp. 1981-1983
NEOPLASIA
Brief report
Both B and T lymphocytes may be clonally involved in
myelofibrosis with myeloid metaplasia
Terra L. Reeder,
Richard J. Bailey,
Gordon W. Dewald, and
Ayalew Tefferi
From the Mayo Clinic, Rochester, MN.
 |
Abstract |
A combination of magnetic cell sorting (MACS) and fluorescent in
situ hybridization (FISH) techniques was used to detect clonal cytogenetic markers in different myeloid and lymphoid cell types of the
peripheral blood from 4 patients with myelofibrosis with myeloid
metaplasia (MMM) that was associated with either a 13q or a 20q
karyotypic abnormality. Interphase cytogenetics studies demonstrated
abnormal clonal FISH signal patterns in neutrophil, myeloid, erythroid,
megakaryocyte, and B- and T-cell preparations in 3 of the 4 patients.
In one patient, FISH results were within normal limits in T cells and
slightly abnormal in B cells. In general, the percentage of abnormal
nuclei was variable in both lymphocyte populations but always higher in
B lymphocytes compared with T lymphocytes. The current study provides
direct evidence for the clonal involvement of both B and T lymphocytes
in MMM. A larger study is needed to clarify the relevance of the
observed interpatient heterogeneity in clonal constitution.
(Blood. 2003;101:1981-1983)
© 2003 by The American Society of Hematology.
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Introduction |
In 1951, Dameshek1 classified
myelofibrosis with myeloid metaplasia (MMM) as a chronic
myeloproliferative disorder (CMPD) along with essential thrombocythemia
(ET), polycythemia vera (PV), and chronic myeloid leukemia (CML)
because of similarities in both clinical and laboratory features.
Between 1967 and 1981, Fialkow and colleauges,2-6 using
glucose-6-phosphate dehydrogenase isoenzyme analysis, showed that CMPDs
are also biologically interrelated on the basis of being clonal stem
cell disorders with involvement of all myeloid cell types
(granulocytes, erythrocytes, platelets, monocytes). In the latter part
of the same period and using similar clonality assays, evidence emerged
that B lymphocytes but not T lymphocytes were clonally involved in CML,
PV, and ET.7-9 More recent studies, based primarily on
X-linked DNA analysis, have supported these early
observations.10-13 Other studies, using different clonal
assays, have suggested monoclonality of T lymphocytes in both MMM
(based on Ras mutational analysis)14 and CML (based on
fluorescent in situ hybridization [FISH] analysis).15 In the current study, we evaluated 4 patients with MMM who had clonal chromosome abnormalities that could be identified by FISH in interphase nuclei in highly purified cell fractions.
| |
Study design |
After approval by the Mayo Clinic institutional review board,
peripheral blood (30 mL) was collected from 4 patients with MMM whose
bone marrow karyotype analysis revealed a deletion of the long arm of
either chromosome 13 (13q) or chromosome 20 (20q). Hypaque density
gradient centrifugation was used to separate out the granulocyte and
mononuclear cell layers (Sigma Diagnostics, St Louis, MO) from each of
the 4 samples. Each mononuclear cell layer was then further cell
fractionated by magnetic cell sorting (MACS; Miltenyi Biotech, Auburn,
CA) by using antibodies that are specific to myeloid
(CD34+), megakaryocyte
(CD61+), and erythroid (CD71+) precursor cells
as well as T (CD3+) and B (CD19+) lymphocytes.
The particular procedure was performed according to the manufacturer's
recommendation. Sample purity in regard to B- and T-cell preparations
was confirmed by flow cytometry (Figure 1). In addition, polymerase
chain reaction (PCR)-based T-cell-receptor (TCR) and immunoglobulin
gene rearrangement studies were performed in lymphocyte-rich,
peripheral blood mononuclear cells from all 4 study patients.
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| Figure 1.
Flow cytometric confirmation of sample purity of B (CD19) and T (CD3)
lymphocyte fractions that are sorted by antibody-linked immunomagnetic
beads from 4 patients with myelofibrosis with myeloid
metaplasia.
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Standard cytospin preparations were made from each of the described
cell preparations and submitted to FISH studies. Each slide was fixed
in methanol/glacial acetic acid (3:1) and air-dried. Slides were
pretreated in 2 × standard saline citrate (SSC) at 37°C for 60 minutes and dehydrated in a series of three 2-minute solutions of 70%,
85%, and 100% ethanol and air-dried. Commercial probes from Vysis
(Downers Grove, IL) for red light scattering index (LSI)
D13S319 and green CEP9 were used together to detect 13q anomalies.
D13S319 hybridizes to 13q14 and CEP9 hybridizes to the alpha Satellite
DNA in the centromere region (9p11-q11) of chromosome 9. Commercial
probes from Vysis for LSI D20S108 (red) and CEP9 (green) were used
together to detect 20q anomalies. D20S108 hybridizes to 20q12. The
cutoff for false-positive nuclei was less than 9.5% for 13q and less
than 11.5% for 20q. Any specimens with percentages of nuclei with
less than the cutoff were classified as "normal." Slides were then
cover-slipped, sealed with rubber cement, denatured at 75°C
for 3 minutes, and allowed to hybridize for 18 to 22 hours at 37°C.
After hybridization, coverslips were removed, slides were washed in
0.4% SSC at 70°C for 2 minutes, and then transferred to 1 ×
phosphate-buffered detergent at room temperature for 1 minute. The
slides were counterstained with a mixture of 5 µL
4'-6'-diamidine-2-phenylindole dihydrochloride (DAPI) and Vectashield
antifade (Vector Labs, Burlingame, CA) at a ratio of 1:10. Cells were
viewed with a fluorescent microscope equipped with a triple-band pass
filter for fluoroisothiocyanate, Texas red, and DAPI (Chromatechnology,
Brattleboro, VT). Each specimen was studied by 2 microscopists
independently. Each microscopist scored 100 consecutive interphase
nuclei, exhibiting 2 clear control signals: the percentages of abnormal
nuclei for the 2 microscopists were averaged to produce a single
percentage of abnormal nuclei for each specimen. Representative B and T
cells were photographed with a computer-based imaging system (Figure
2).
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| Figure 2.
Fluorescent in situ hybridization studies of T and B lymphocytes.
T (A) and B (B) lymphocytes from 2 patients with myelofibrosis with
myeloid metaplasia that exhibit a single orange signal revealing a
deletion of the long arm of either chromosomes 20 (20q) or 13 (13q), respectively, by fluorescent in situ hybridization studies.
Both green signals of the control probe (CEP9) are visible in
each figure. Original magnification,
× 100.
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| |
Results and discussion |
The 4 study patients (3 men and 1 woman) with MMM had bone marrow
chromosomal abnormalities including either 13q [del(13)(q12q14); del(13)(q12q21.2)] seen in 2 patients or 20q [del(20)(q13.1)] seen
in the other 2 patients. Three of the 4 patients had either 13q or
20q in all metaphases (Table 1). In the
fourth patient, 20q was carried by 16 of 21 metaphases. In 3 of the 4 patients, FISH analysis revealed an abnormal signal pattern in
neutrophils, erythroid (CD71+), megakaryocyte
(CD61+), and myeloid (CD34+) cells as well as B
(CD19+) and T (CD3+) lymphocytes (Table 1). The
results in the 4th patient were similar, except that the T-cell
population FISH results were within normal limits (Table 1). We
observed substantial interpatient heterogeneity in the percentage of
abnormal nuclei in both B (13%-96%) and T lymphocytes (6%-57%) as
well as CD34+ cells (36%-87%) (Table 1). Among the 3 patients with only abnormal metaphases, the highest percentage of
abnormal nuclei was demonstrated by neutrophils (82%-98%),
megakaryocyte (CD61+) (75%-94%), and erythroid
(CD71+) (78%-88%) cell types.
View this table:
[in this window]
[in a new window]
|
Table 1.
Percentage of abnormal nuclei in each cell lineage of 4 patients with myelofibrosis with myeloid metaplasia studied with
fluorescent in situ hybridization using probes to detect 13q and
20q
|
|
Information from X chromosome-based clonality assays may not be
totally adequate because of the occurrence of both excessive Lyonization and acquired skewing patterns of hematopoiesis in normal
elderly women.16-18 Therefore, the application of
alternative methods is essential for further clarification of clonal
constitution in myeloid disorders. In this regard,
karyotype,19,20 FISH,15,21 or Ras mutational
analysis14 have been applied to either fractionated cell
populations or in vitro progenitor colonies. In most, but not
all,14,15 instances, the results have been consistent with those derived from X-linked clonality assays.
The current clonality study was FISH based and clearly indicates that
both B and T lymphocytes may be clonally involved in MMM. The absence
of clonal immunoglobulin and TCR gene rearrangements, in
lymphocyte-rich cell fractions from our patients, is consistent with
the current assumption that the disease-initiating mutation in MMM and
related disorders occurs before the earliest biologic events that are
associated with lymphocyte commitment. In addition, the study suggests
substantial interpatient heterogeneity in clonal constitution of
myeloid progenitor cells (CD34+) as well as both B and T
lymphocytes. However, this variation in the expression of a
FISH-detected "clonal" marker in a selected cell population may
reflect dynamic differences in clonal evolution rather than a
difference in the distribution of the original disease clone.22 In other words, a FISH-detected lesion may
represent a cytogenetic subclone, and the absence of such a marker does not necessarily imply nonclonality. Similarly, the observed
heterogeneity in the degree of clonal signal distribution may apply
only to cytogenetic subclones and not to the original disease clone.
Nevertheless, the current study raises questions about the validity of
using T cells as controls in biologic studies of myeloid disorders.
| |
Footnotes |
Submitted August 2, 2002; accepted October 11, 2002.
Prepublished
online as Blood First Edition Paper, October 24, 2002;
DOI 10.1182/blood-2002-07-2341.
Supported by Mayo Clinic Hematology Research.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Ayalew Tefferi, Division of Hematology, Mayo
Clinic, 200 First St SW, Rochester, MN 55905; e-mail:
tefferi.ayalew{at}mayo.edu.
| |
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Abstract
Introduction