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Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 771-772
BRIEF REPORT
Immunohistochemical staining for megakaryocyte c-mpl may
complement morphologic distinction between polycythemia vera and
secondary erythrocytosis
Ayalew Tefferi,
Soo-Young Yoon, and
Chin-Yang Li
From the Division of Hematology and Internal Medicine and the
Division of Hematopathology, Mayo Clinic and Mayo Foundation,
Rochester, MN.
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Abstract |
Recent studies have shown decreased megakaryocyte expression of the
thrombopoietin receptor (c-mpl) in patients with polycythemia vera (PV) but not in those with reactive erythrocytosis. We examined the diagnostic utility of this observation in 22 patients with PV, 7 patients with secondary erythrocytosis (SE), and 10 normal controls.
Commercial antibodies against c-mpl were used with standard immunoperoxidase methods. Megakaryocyte c-mpl staining
intensity was uniformly moderate-to-strong in the healthy controls and
in all the patients with SE. In contrast, staining intensity in 9 patients with PV (41%) was uniformly weak. Furthermore, in 12 of the
remaining 13 patients with PV, the c-mpl staining pattern in
each case was heterogeneous and was associated with weak staining intensity in more than 20% of the megakaryocyte population. These preliminary data suggest that c-mpl immunostains may complement bone marrow histopathology in distinguishing PV from nonclonal causes
of erythrocytosis.
(Blood. 2000;96:771-772)
© 2000 by The American Society of Hematology.
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Introduction |
The use of disease-specific biologic assays has
resulted in the identification of patients with polycythemia vera (PV)
who do not strictly fulfill the Polycythemia Vera Study Group (PVSG) diagnostic criteria.1-3 In this regard, both serum
erythropoietin and endogenous erythroid colony assays have been shown
to be diagnostically useful in distinguishing PV from secondary
erythrocytosis (SE).4,5 However, both of these assays have
substantial limitations. For example, although a decreased or increased
serum erythropoietin level is consistent with a diagnosis of PV and SE,
respectively, a "normal" level does not exclude either
diagnosis.6,7 Similarly, the diagnostic value of an
endogenous erythroid colony growth assay is hampered by the lack of
widespread expertise in performing the test and interpreting the
results.5
Therefore, the development of additional biologic assays that are both
practical and relatively specific may not only help in distinguishing
between PV and SE but may also allow detection of cases of PV that do
not manifest the full complement of the PVSG criteria. In a recent
study, platelet expression of the thrombopoietin receptor
(c-mpl) was shown to be reduced in all 34 patients with PV who
were tested and in none of the 14 patients with SE.8 Similarly, decreased megakaryocyte c-mpl expression was
reported in 10 patients with PV. In the current study, we evaluated the diagnostic value of c-mpl-based bone marrow
immunohistochemical stains in 22 patients with PV, 7 patients with SE,
and 10 normal controls.
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Study design |
Conventional criteria were used to identify patients with
PV.9 The comparative groups included both normal controls
and patients with SE. Immunohistochemical staining of c-mpl, in
paraffin-embedded bone marrow sections, was accomplished by an
immunoperoxidase method using avidin-biotin complexes and a mouse
monoclonal anti-human c-mpl antibody (Genzyme, Cambridge,
MA). Bone marrow section slides were deparaffinized in
xylene, hydrated in sequential gradients of ethanol, and pretreated
with heated ethylenediamine tetraacetic acid (1 mmol/L, pH 8.0) for 30 minutes. This was followed by sequential incubation in 1:200 dilution
of c-mpl antibody for 1 hour, biotinylated goat
anti-mouse antibodies (Dako, Denmark) for 30 minutes, and avidin-biotin complex reagent (Vecta Stain, Vector
Laboratories, Burlingame, CA) for 30 minutes. After
induction of the color reaction with 3-amino-9-ethylcarbazole substrate
solution (Sigma, St Louis, MO), the slides were
counterstained with hematoxylin.
The staining intensity (weak vs moderate vs strong) and pattern
(uniform vs heterogeneous) of megakaryocyte c-mpl expression were semiquantitatively and independently evaluated by all the authors.
In addition, 2 of the authors (S.Y.Y. and C.Y.L.) were blinded to the
clinical diagnosis during the interpretation of the immunostains. In an
individual patient, the staining pattern was categorized as being
uniform if more than 80% of the megakaryocytes displayed similar
staining intensity. Otherwise, the staining pattern was classified as
being heterogeneous and the associated staining intensity connotation
was based on majority. The histologic assessment of megakaryocyte
number and morphology was reviewed jointly by 2 of the
authors (S.Y.Y. and C.Y.L.). Statistical calculations were
performed by using StatView software (Version 5.0.1, SAS Institute,
Cary, NC).
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Results and discussion |
Bone marrow c-mpl studies were performed on bone marrow
trephine biopsy specimens of 22 patients with PV, 7 with SE, and 10 normal controls. In the patients with PV, the immunohistochemical studies were performed either at diagnosis (9 patients) or later in the
course of the disease. At the time of the study, 15 patients were
chemotherapy naïve, 9 had a history of thrombosis, and 6 were
showing early signs of transformation of their disease into myelofibrosis with myeloid metaplasia. The causes of SE were central hypoxia (3 cases), central hypoxia and diabetes insipidus (1 case), high-oxygen-affinity hemoglobinopathy (1 case), and apparent
erythrocytosis (2 cases). The median hemoglobin value in patients with
SE was 18.7 g/dL. As outlined in Table 1,
and consistent with previous studies,10 bone marrow
histopathologic differences between PV and SE were readily apparent.
In general, staining intensity of megakaryocyte c-mpl was
uniformly moderate to strong in both normal controls and all the patients with SE (Figure 1A and B, Table
2). In contrast, weak staining intensity
was demonstrated in at least 20% of the megakaryocyte population in 21 of the 22 patients with PV. In 9 of these 22 patients (41%), more than
80% of the megakaryocytes displayed weak staining intensity (Figure
1C, Table 2). In 12 of the 13 remaining patients with PV, the staining
pattern was heterogeneous (Figure 1D, Table 2). One patient with PV
showed moderate staining intensity in a uniform fashion. These
variations in staining intensity and pattern did not correlate with the
duration or stage of the disease and were also not influenced by the
presence of chemotherapy (data not shown).
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| Fig 1.
Bone marrow immunohistochemical stains for megakaryocyte
c-mpl.
(A) Uniformly strong staining in secondary erythrocytosis; (B)
uniformly moderate staining in a normal control; (C) uniformly weak
staining in a patient with polycythemia vera; (D) heterogeneous
staining in another patient with polycythemia vera.
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View this table:
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Table 2.
Comparison of bone marrow c-mpl staining pattern
and intensity among 22 patients with polycythemia vera, 7 patients with
secondary erythrocytosis, and 10 normal controls
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These observations are mostly consistent with a recent study that
demonstrated reduced c-mpl expression by platelets and
megakaryocytes in patients with PV, but not in those with
SE.8 However, this study suggests that megakaryocyte
c-mpl expression, both in a particular patient and between
patients, may show substantial heterogeneity. The differences between
the 2 studies may be due to differences in antibody preparations,
although the mouse monoclonal antibody we used is also directed against
the extracellular domain of the c-mpl protein.11 On
the other hand, the observed interpatient and intrapatient variations
in staining intensity may be related to differences in the content of
an underglycosylated c-mpl isoform that has been reported to
exist in patients with PV.11 Interpatient variability in
platelet c-mpl expression has previously been reported in
patients with ET.12
It should be recognized that the majority of patients with PV present
with clinically evident disease and the diagnosis may not require the
use of sophisticated tests. In cases that are difficult to diagnose,
however, bone marrow immunohistochemical staining for c-mpl may
complement the diagnostic value of bone marrow histopathology and
obviate in vitro endogenous erythroid colony assays.
| |
Footnotes |
Submitted February 2, 2000; accepted March 14, 2000.
Reprints: Ayalew Tefferi, Division of Hematology and
Internal Medicine, Mayo Clinic, 200 First St SW, Rochester, MN 55905.
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.
| |
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Abstract
Introduction