Blood, 15 July 2002, Vol. 100, No. 2, pp. 714-717
BRIEF REPORT
The expression pattern of c-mpl in megakaryocytes
correlates with thrombotic risk in essential thrombocythemia
Luciana Teofili,
Francesco Pierconti,
Annalaura Di
Febo,
Nicola Maggiano,
Nicola Vianelli,
Stefano Ascani,
Elena Rossi,
Stefano Pileri,
Giuseppe Leone,
Luigi Maria Larocca, and
Valerio De
Stefano
From the Departments of Hematology and Pathology,
Catholic University, Rome, Italy; and the Institute of Hematology
Seragnoli, University of Bologna, Bologna, Italy.
 |
Abstract |
Using immunohistochemistry, we investigated the expression of
c-mpl in bone marrow megakaryocytes of 88 patients with
essential thrombocythemia (ET), 6 patients with secondary
thrombocytosis (ST), and 20 patients with lymphoma (controls).
Considering both the pattern of expression and the staining intensity,
we identified a uniform and a heterogeneous pattern of c-mpl
expression. The uniform pattern was found in all the controls,
all the patients with ST, and 28 of the patients with ET, with a strong
staining intensity observed in most megakaryocytes (> 80%). In
contrast, c-mpl expression was heterogeneous in 60 patients
with ET, 18 of whom (30%) presented with thrombosis at diagnosis, a
significant difference from patients with a uniform
c-mpl pattern (2 of 28; 7%; P = .026).
In particular, the overrepresentation of thrombotic complications in
patients with a heterogeneous c-mpl expression pattern was
found mainly among patients with a significant percentage (10% to
40%) of weakly stained or c-mpl-negative megakaryocytes (heterogeneous-weak pattern; 13 of 30; 43%;
P = .002). Accordingly, this pattern was
associated with a 6.1-fold increased risk of thrombosis compared with
that of patients with a uniform c-mpl pattern. In
conclusion, the presence of a heterogeneous pattern of
c-mpl distribution in bone marrow megakaryocytes could be a useful diagnostic criterion in the differential diagnosis of
thrombocytosis. Furthermore, detection of a significant percentage of
weakly stained or c-mpl-negative megakaryocytes can
identify patients with a higher risk of thrombosis.
(Blood. 2002;100:714-717)
© 2002 by The American Society of Hematology.
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Introduction |
Thrombopoietin (TPO), the primary regulator
of megakaryocyte and platelet development, acts by stimulating
megakaryocyte growth from bone marrow (BM) progenitors, by inducing
terminal differentiation of immature megakaryocytes, and by supporting
production of functional platelets.1,2 All these effects
are mediated by the binding of TPO to a cell-surface receptor encoded
by the c-mpl proto-oncogene, which is expressed by
CD34+ progenitors, megakaryocytes, and circulating
platelets.3,4 Impaired expression of c-mpl in
c-mpl-deficient mice invariably induces a marked
thrombocytopenia due to reduced production of BM
megakaryocytes.5
In contrast, among hematologic diseases, reduced c-mpl
expression has been found in chronic myeloproliferative disorders
(CMDs) typically sharing an increase in platelet production (reviewed by Tefferi6). In particular, platelets isolated from
patients with polycythemia vera (PV) and idiopathic myelofibrosis
showed impaired expression and activation of c-mpl after
exposure to TPO,7-9 associated with decreased
immunostaining for c-mpl in BM
megakaryocytes.9,10 Furthermore, several studies found that in some cases of essential thrombocythemia (ET), expression of
c-mpl on platelets is also reduced at both the protein and the RNA level.11-13 Moreover, a study in 9 patients with
ET showed that the immunostaining of megakaryocytes for
c-mpl was highly heterogeneous.14 In the
current study, we used immunohistochemistry to investigate the pattern
of megakaryocyte expression of c-mpl in patients with ET and
patients with secondary thrombocytosis (ST).
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Study design |
The study was carried out retrospectively in BM biopsy specimens
collected from 88 patients with ET treated nonconsecutively between May
1993 and June 2001 at 2 different Italian hematology centers: the
Division of Hematology, Catholic University of Rome (44 patients), and
the Institute of Hematology Seragnoli, University of Bologna (44 patients). All patients with ET were studied at the time of diagnosis,
except for one patient with a diagnosis of latent chronic
myeloproliferative disease15,16 who had thrombosis of
portal and mesenteric veins, normal peripheral blood counts, and growth
of endogenous erythroid colonies (EECs). All patients with ET
fulfilled the conventional diagnostic criteria17 at the
time of BM biopsy, whereas the patient with growth of EECs had onset of
an overt form of ET 5 years later. Clinical and hematologic characteristics of the patients with ET are shown in Table
1.
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Table 1.
Clinical and laboratory characteristics in patients with
essential thrombocythemia, according to pattern of c-mpl
expression
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Twenty-one patients (10 men and 11 women; median age, 47 years; range,
25 to 79 years) had a previous history of thrombosis: 5 had an ischemic
stroke, 5 a myocardial infarction, 5 portal and mesenteric vein
thrombosis, 2 hepatic vein thrombosis, 2 cerebral vein thrombosis, and
2 deep venous thrombosis of the legs. All the thrombotic events had
been proven by use of objective criteria. All patients had the
thrombotic event at the time of diagnosis or during the previous 3 months, except for one male patient who had a myocardial infarction 12 years earlier. This event was considered not related to the hematologic
disease and was not included in the calculation of estimated thrombotic
risk. We also studied 6 patients with ST (2 patients with
infectious disease and 4 patients who had undergone splenectomy) and 20 patients undergoing staging procedures for Hodgkin and non-Hodgkin
lymphomas with no evidence of BM involvement, who were considered controls.
BM specimens were obtained by using standard procedures and embedded in
paraffin. Immunohistochemical analysis was performed with the
avidin-biotin-peroxidase complex method. Endogenous biotin sites were
blocked by sequential incubation with avidin-biotin solutions (ABC
ELITE detection system; Vector Laboratories, Burlingame, CA).
Expression of c-mpl was investigated by using a mouse
monoclonal antibody antihuman c-mpl (R&D Systems,
Minneapolis, MN) after a previous step of antigen retrieval in a
microwave oven for 30 minutes in EDTA buffer (1 mM; pH 8), at 250 W. Slides were then incubated for 1 hour at a 1:200 dilution of
c-mpl antibody. Hydrogen peroxide, serum biotinylated
immunoglobulins, and avidin-biotin complexes were used according to the
manufacturer's instructions (Dako LSAB; Dakopatts, Golstrup, Denmark).
After induction of the color reaction with freshly made
diaminobenzidine solution (Dakopatts), slides were counterstained with hematoxylin.
The c-mpl staining intensity (weak versus moderate versus
strong) was semiquantitatively established by 2 pathologists (F.P. and
L.M.L.) in assessment of control biopsy specimens. These investigators independently evaluated the staining intensity and the pattern (uniform
versus heterogeneous) of megakaryocyte c-mpl expression in
patients with ET and ST while blinded to the clinical diagnosis of
individual patients. Interobserver variation for each sample was less than 5% and was omitted. All megakaryocytes present in each
BM biopsy specimen were analyzed for c-mpl staining
intensity, and only specimens containing at least 100 megakaryocytes
were included in the study. Strongly stained plasma cells were used to
control the quality of the staining. Statistical analysis of the data
included use of the Mann-Whitney U test or Fisher exact test
for direct comparisons and the Kruskal-Wallis test or 
2
test for multiple comparisons, where appropriate.
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Results and discussion |
We identified 2 patterns of expression of c-mpl: a
uniform pattern (> 80% strongly stained megakaryocytes, with moderate
to weak immunoreactivity in the remaining megakaryocytes) and a
heterogeneous pattern (< 80% strongly stained megakaryocytes and more
than 20% moderately to weakly stained megakaryocytes). In all BM
biopsy specimens obtained from controls and patients with ST,
c-mpl was expressed uniformly and the intensity of the
staining was strong in most megakaryocytes (> 80%; Table 1 and Figure
1A). In contrast, only 28 of the 88 patients with ET had a uniformly strong expression of c-mpl.
The remaining 60 patients had a heterogeneous pattern of
c-mpl expression. In 30 of them, strongly expressing
c-mpl megakaryocytes were observed together with
megakaryocytes showing moderate c-mpl expression
(heterogeneous-moderate pattern; > 20%; Figure 1B). In the other 30 patients, strongly and moderately stained megakaryocytes were
associated with a consistent amount of weakly stained or
c-mpl-negative megakaryocytes (heterogeneous-weak pattern;
10% to 40% of the total; Table 1 and Figure 1C and 1D). The pattern
of expression of c-mpl was not significantly related to the
presence of BM hypercellularity (Table 1). These findings are in
agreement with those of Yoon et al,14 who described a heterogeneous distribution of c-mpl in ET.
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| Figure 1.
Expression of c-mpl in essential thrombocythemia.
(A) All megakaryocytes show a uniform, strong staining intensity
(× 320). The inset shows a higher magnification of a strongly stained
megakaryocyte (× 1000). (B) Heterogeneous pattern of c-mpl
expression with coexistence of strongly positive megakaryocytes and a
significant percentage of megakaryocytes showing a moderate staining
intensity (× 250). Some strongly stained plasma cells are also
present. The inset shows a higher magnification of a moderately stained
megakaryocyte (× 1000). (C) Heterogeneous pattern of c-mpl
staining with moderately positive megakaryocytes together with several
weakly stained megakaryocytes (× 250). Several plasma cells show
strong c-mpl positivity. The inset shows a higher
magnification of a cluster of weakly stained megakaryocytes (× 1000).
(D) Heterogeneous pattern of c-mpl expression characterized
by the presence of several c-mpl-negative megakaryocytes
together with some moderately stained megakaryocytes and a few strongly
stained plasma cells (× 250). The inset shows a higher magnification
of a c-mpl-negative megakaryocyte (× 1000)
(avidin-biotin-peroxidase-complex method in paraffin section
lightly counterstained with hematoxylin).
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We then compared the clinical and laboratory findings in patients with
a uniform c-mpl pattern with those in patients with a
heterogeneous pattern. No significant differences were found with
regard to hemoglobin level (P = .81), hematocrit value
(P = .77), platelet count (P = .08), sex
distribution (P = .64), age (P = .85), or
presence of splenomegaly (P = .20). Patients with a
heterogeneous pattern of c-mpl-expression had significantly higher white blood cell (WBC) counts than other patients
(P = .02). Moreover, an increased rate of previous
thrombotic events was found in patients with heterogeneous c-mpl
expression (30%) compared with patients with uniform c-mpl
expression (7%; P = .026).
In an analysis considering the 2 subgroups of patients with
heterogeneous expression (heterogeneous-moderate and
heterogeneous-weak) in comparison to the patients with a uniform
pattern, no differences in sex distribution, age, hemoglobin level,
hematocrit value, platelet count, or presence of splenomegaly were
found among these 3 different groups (Table 1). However, only patients
with a heterogeneous-weak pattern had a significantly higher incidence
of thrombosis (43%) than patients with a uniform
c-mpl-expression pattern (P = .002). The
relative risk of thrombosis associated with the presence of a
heterogeneous-weak pattern was 6.1-fold higher (95% confidence interval, 1.5-24.5) than that associated with a uniform
c-mpl pattern. Patients with a heterogeneous-moderate
c-mpl pattern had a higher rate of thrombotic complications
than those with a uniform pattern, although the difference was not
significant (P = .424). Compared with patients with a
uniform c-mpl expression pattern, WBC counts were
significantly higher in patients with a heterogeneous-moderate pattern
(P = .050) as well as in patients with a
heterogeneous-weak pattern (P = .025).
Several data support the idea that TPO potentiates platelet activation
by means of a variety of stimuli, including thrombin, adenosine
diphosphate, collagen, and adrenaline18; thus, the association between a megakaryocyte population that is not responsive to TPO and an increased risk of thrombosis is rather
unexpected. Nevertheless, it can be hypothesized that a
decreased uptake of TPO by megakaryocytes with impaired c-mpl
expression could result in a increase in TPO availability for
stimulation and activation of the remaining, normally
c-mpl-expressing platelets. In agreement with this
hypothesis, the increased WBC count found in these patients could have
resulted from a greater expansion of the granulocyte-macrophage progenitors due to the increased TPO availability.19
Studies investigating the presence of monoclonal and polyclonal forms
of ET suggested that, in contrast to the other CMDs, ET is a
heterogeneous disease.20-22 Our finding of 2 distinct
patterns of c-mpl expression in BM megakaryocytes from
patients with ET seems to support this concept. Thus, although the
finding of a uniformly strong expression of c-mpl does not
exclude the diagnosis of ET, the finding of a heterogeneous pattern of
c-mpl distribution could be a useful criterion for
distinguishing ET from reactive thrombocytosis.
Although thrombosis is considered the most important complication of
ET, the only currently established risk factors for a thrombotic event
are retrospective, such as a previous history of thrombosis, advanced
age, and the duration of thrombocytosis23; variables
predictive of thrombotic complications are lacking. Our study was
retrospective, but the possible bias resulting from inclusion of
patients with severe thrombotic manifestations did not seem to affect
our results. In fact, in our series, the overall rate of
thrombotic complications was 20%, a proportion in good agreement with
current estimates in patients with ET.23 In addition, the
evaluation of the c-mpl pattern was carried out by
investigators who were blinded with respect to clinical history.
Finally, patients with thrombosis and a heterogeneous-weak
c-mpl pattern had not been preferentially referred in
comparison with other patients.
We conclude that the presence of a heterogeneous-weak pattern of
c-mpl expression in BM biopsy specimens could be a useful tool for identifying patients with ET with a higher risk of
thrombosis at the time of diagnosis. Moreover, detection of an abnormal
pattern of c-mpl expression may also identify latent forms
of CMD in patients not fulfilling conventional diagnostic criteria for
ET or PV, as has already been reported for the EEC
assay.15,16
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Footnotes |
Submitted August 2, 2001; accepted March 7, 2002.
Supported in part by grants from Università Cattolica del Sacro
Cuore, Milan, Italy; Ministero della Università e della Ricerca
Scientifica, Rome, Italy; and Associazione Italiana per la Ricerca sul
Cancro, Milan, Italy.
L.T. and F.P. contributed equally to this work.
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: Luciana Teofili, Istituto di Ematologia,
Università Cattolica, Largo Gemelli 8, 00168 Rome, Italy; e-mail:
lteofili{at}rm.unicatt.it.
| |
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Abstract
Introduction
