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Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 417-424
By
From the Department of Haematology, University College London Medical
School, London, UK.
Essential thrombocythemia (ET) is traditionally considered to be a
clonal disorder. No specific karyotypic abnormalities have been
described, but the demonstration of clonality using X-chromosome inactivation patterns (XCIPs) has been used to differentiate ET from a
non-clonal reactive thrombocytosis. However, these assays may be
difficult to interpret, and contradictory results have been reported.
We have studied 46 females with a diagnosis of ET according to the
Polycythemia Vera Study Group (PVSG) criteria. XCIP results in 23 patients (50%) were uninterpretable due to either constitutive or
possible acquired age-related skewing. Monoclonal myelopoiesis could be
definitively shown in only 10 patients. Thirteen patients had
polyclonal myelopoiesis, and in 8, it was possible to exclude clonal
restriction to the megakaryocytic lineage. Furthermore, there was no
evidence of clonal progenitors in purified
CD34+CD33
ESSENTIAL thrombocythemia (ET) is
characterized by a sustained thrombocytosis with a tendency to
thrombosis and hemorrhage. It is predominantly a disease of the
elderly, with a median age at presentation of 60 to 70 years, although
since the introduction of routine assessment of platelet counts, more
patients are being diagnosed at an earlier age.1 The
disorder is usually considered to be a clonal disease arising in a
multipotent stem cell and to be related to the other clonal
myeloproliferative disorders, such as polycythemia vera (PV) and
chronic myeloid leukemia (CML). The criteria proposed by the
Polycythemia Vera Study Group (PVSG) remain the gold standard for
establishing a diagnosis of ET, but these are largely guidelines for
the exclusion of other conditions associated with a
thrombocytosis.2 PV is excluded either by a hemoglobin of
less than 13 g/dL or a normal red blood cell mass in the presence of
normal iron stores; myelofibrosis is excluded if collagen fibrosis in
the trephine biopsy is less than 33% with absence of
leucoerythroblastic features on the blood film; and CML is excluded by
lack of cytogenetic or molecular evidence of the bcr/abl
rearrangement. A small but significant proportion of patients, 3% to
4%, ultimately transform to an acute leukemic phase, which is in
accord with the assumption that ET is a clonal disorder of a
hemopoietic stem cell, although this is frequently related to the
therapy administered, including radiophosphorous and alkylating
agents.1 A recent report has also suggested that the
ribonucleotide reductase inhibitor hydroxyurea may cause secondary
leukemia in patients with ET.3
The major diagnostic difficulty encountered is in discriminating ET
from an occult cause of a reactive and persistent thrombocytosis (RT).
A number of different approaches have been used to try and identify
diagnostic features that are specific for ET and that enable exclusion
from RT. A variety of platelet defects have been documented, but they
are not consistently found in all ET patients and are not sufficiently
specific to be of diagnostic use.4 Clonogenic assays have
also attracted attention, because in some patients with ET and other
myeloproliferative disorders, a proportion of hematopoietic progenitors
show growth factor independence. However, there are problems of
interpretation with these assays, because a wide variety of culture
conditions have been used and spontaneous megakaryocyte colonies have
also been found in patients with RT.5 No characteristic
karyotypic abnormalities have been associated with ET at diagnosis,
although a recent report has shown an increased incidence of
abnormalities involving 17p in patients treated with
hydroxyurea.3
A number of groups have shown myeloid clonality using X-linked
polymorphic markers in female ET patients. The earliest reports used
isoenzymes of the protein glucose-6-phosphate dehydrogenase (G6PD), but
the number of patients studied was small due to the low incidence of
heterozygosity.6 Later studies used differential DNA
methylation patterns of active and inactive X-chromosomes of genes,
such as phosphoglycerate kinase (PGK), hypoxanthine phosphoribosyl
transferase (HPRT), and the human androgen receptor (HUMARA).7-10 The most recent studies have used expression
of transcripts in RNA of three polymorphic genes,
iduronate-2-sulphatase (IDS), palmitoylated membrane protein p55, and
G6PD.9
These results must be interpreted with appropriate reference to both
the individuals' constitutive X-chromosome inactivation pattern (XCIP)
and their age. Approximately 20% to 25% of hematologically normal
females have a constitutively imbalanced or skewed XCIP with greater
than 75% expression of one allele.11 An imbalanced pattern
therefore can only be interpreted as clonal if control tissue from the
same individual shows a balanced or significantly different pattern. T
lymphocytes are thought to be the most suitable control for disorders
of myeloid cell origin, because they originate from the same
pluripotent stem cell, and in younger hematologically normal
individuals, they have the same XCIP.12 However, studies have also shown that there is an increased incidence of skewing in
myeloid cells of the elderly,13,14 which may not be
reflected in T-lymphocyte XCIPs.10,11 In our own study, for
example, an extremely skewed pattern with greater than 90% expression
of one allele was found in the neutrophils of 33% and T lymphocytes of
9% of hematologically normal females more than 75 years of age, but in
only 3% of younger individuals.11 In the few studies of ET
patients in which T-lymphocyte XCIPs have been compared with neutrophil
patterns, the results have been conflicting. Tsukamoto et
al15 showed monoclonal XCIPs in neutrophils of all 13 patients in their study, suggesting that ET is almost invariably a
clonal disorder, although we would only consider 6 of these patients to
be assessable because of advanced age or constitutive skewing of the T
cells. In a study of 46 patients by El-Kassar et al,9 the
majority seemed to have monoclonal neutrophils (61%), but 14 patients
had polyclonal patterns in both neutrophils and T lymphocytes. Ten of
these 14 patients could be studied using RNA polymorphisms. Platelet
polyclonality was confirmed in 7 patients, and monoclonal platelets
were found in 3 patients, these latter results suggesting that ET could
sometimes be restricted to the megakaryocytic line. Such lineage
restriction is in marked contrast to other clonal myeloproliferative
disorders.16
In the current study, we have sought to determine the frequency of
polyclonal myelopoiesis in female patients with a diagnosis of ET
fulfilling the PVSG criteria and to determine in such patients the
incidence of megakaryocyte/platelet-restricted clonality. In addition,
we have analyzed the precursor/progenitor cell fractions of the bone
marrow from 3 patients to ascertain whether the clonal status of these
populations differs from the neutrophils in the peripheral blood.
Finally, we have compared the clinical features of those patients with
definite clonal and polyclonal hemopoiesis to determine the clinical
relevance of clonality studies.
Patients
Controls
Sample Preparation
Peripheral blood.
Peripheral blood collected into EDTA was centrifuged at 180g
for 15 minutes, then the upper two thirds of the plasma was harvested as platelet-rich plasma and washed twice at 2,000g with calcium and magnesium-free phosphate-buffered saline (PBS)
containing 10 mmol/L EDTA. The remaining sample was resuspended to the
original volume with PBS and sedimented with 10% dextran (final
concentration, 1%) to reduce red blood cell contamination, and then
the supernatant was spun through Ficoll (Ficoll Hypaque; Pharmacia
Biotech, Uppsala, Sweden). T lymphocytes were purified from the
mononuclear layer using anti-CD3-coated magnetic beads (Dynal, New
Ferry, UK). Additional red blood cells were removed from the neutrophil
fraction by hypotonic lysis. Purity of the CD3+ cells was
determined by morphological examination and neutrophil and platelet
fractions on a whole blood cell analyzer (Sysmex, Milton Keynes, UK).
Bone marrow.
Bone marrow samples were collected into minimum essential medium
(MEM; GIBCO-BRL, Paisley, UK) containing 20 U/mL heparin and 1 mg/mL EDTA and incubated with a 1% solution of Red-Out, a
glycophorin antibody (Robbins Scientific, Sunnyvale, CA), at room
temperature for 10 minutes. Mononuclear cells were prepared by Ficoll
density gradient centrifugation, washed twice with PBS containing 1%
albumin and 1 mg/mL EDTA, and then incubated with anti-CD34 multisort
beads (Miltenyi Biotech, Camberly, UK) at 4°C for 30 minutes.
CD34+ cells were selected on a "Variomacs" column
(Miltenyi Biotech), and the purity was checked by
fluorescence-activated cell sorter (FACS) and alkaline
phosphatase-anti-alkaline phosphatase (APAAP) analysis.17 The anti-CD34-coated beads were removed with a
release reagent (Miltenyi Biotech), and the cells were washed then
further separated into CD33 DNA and RNA.
DNA was prepared from separated cells using detergent
lysis18 and RNA with Trizol (GIBCO, Grand Island,
NY).
XCIP Analysis
DNA.
Samples were screened for heterozygosity of the PGK and HUMARA genes,
and clonal analysis was performed as described previously19 (Harrison et al, manuscript in press). Samples were also
screened for heterozygosity in the IDS, p55, and G6PD genes using
mismatched primers. Details of the primers used, their
location, fragment sizes, and restriction enzyme digests are given in
Table 2. Approximately 1 µg DNA was added
to a reaction mix containing 1× Taq polymerase buffer (50 mmol/L KCl, 10 mmol/L Tris-HCl, pH 9.0, 0.1% Triton X-100), 2.25 mmol/L (IDS and p55) or 3 mmol/L (G6PD) MgCl2, 0.2 mmol/L
each dNTP (all from Promega, Madison, WI), and 10 pmol each primer, in
a total volume of 19 µL. The mix was heated to 95°C for 5 minutes,
held at 85°C while 1 µL containing 0.5 U Taq polymerase was
added, and then 35 cycles of 95°C for 30 seconds, 66°C (IDS) or
68°C (p55 and G6PD) for 30 seconds, and 72°C for 30 seconds were
performed, followed by a final extension at 72°C for 5 minutes.
Polymerase chain reaction (PCR) products were digested with the appropriate restriction enzyme for at least 4 hours, and then
electrophoresed through 3% agarose in 1× Tris-Borate-EDTA (TBE) and visualized by ethidium bromide staining under
UV light.
RNA.
Approximately 1 µg RNA was incubated with 250 ng oligo
(dT)15 in a total volume of 12 µL at 65°C
for 5 minutes, and then cooled to room temperature for 10 minutes.
Reaction mix was added to give a final concentration of 1×
Taq polymerase buffer, 5.25 mmol/L MgCl2, 1 mmol/L
each dNTP, 20 U RNAse inhibitor, and 5 U avian myeloblastosis virus
(AMV) reverse transcriptase (all from Promega), and the
mix was incubated at 42°C for 60 minutes followed by 95°C for 5 minutes. Complementary DNA (4 µL) was used in a PCR reaction as
described above but with the following modifications: the primers used
were all located in exons and differed from the DNA primers for IDS and
p55 analysis (Table 2); final MgCl2 concentrations were
2.25 mmol/L (IDS and p55) or 3 mmol/L (G6PD); 1 pmol 32P
end-labeled primer (IDS/UR, p55/DR, or G6PD/U) was added
with the Taq polymerase; 25 cycles of amplification were
performed; and annealing temperatures were 66°C (IDS), 64°C (p55),
or 68°C (G6PD). PCR products (10 µL for IDS and G6PD and 5 µL for
p55) were incubated with the appropriate restriction enzyme (Table 2)
for at least 4 hours, and then electrophoresed through a nondenaturing polyacrylamide gel (10% for IDS and G6PD, 6% for p55; 10 × 8 cm, cross-linker ratio 19:1, 1× TBE). The gel was dried and exposed to
Hyperfilm-MP (Amersham Life Science, Little Chalfont, UK). Autoradiographic signals were quantified by scanning densitometry (Hoefer Instruments, San Francisco, CA) and reported as the percentage of expression of the lower allele. Each sample was analyzed in duplicate, and results were expressed as a mean of the two values. Results were considered to be significantly different if there was
greater than 20% difference between the two values, the limit of our
technical variation as determined in previous studies19 (Harrison et al, manuscript in press).
XCIP results were obtained on neutrophils and T cells from a total of
46 ET patients. The mean purity of neutrophil preparations was 91%
(range, 75% to 99%), and the T-cell fraction contained an average of
97% lymphocytes (range, 82% to 100%). DNA was analyzed in 43 patients using either HUMARA (n = 38), PGK (n = 3), or
both (n = 2). RNA from neutrophils, T cells, and platelets was
analyzed in 21 patients using either IDS (n = 13), p55 (n = 4), or
G6PD (n = 4). Results were also obtained on neutrophils and T cells from 13 RT patients using DNA (HUMARA, n = 10; PGK and HUMARA, n = 3) and from 9 of these patients using IDS-RNA analysis. Myeloid progenitor/precursor populations purified from the bone marrow were
analyzed in three ET patients, and purified megakaryocytes from 1 of these patients were analyzed.
Comparison of DNA and RNA XCIP Results
RT Patients
ET Patients On the basis of the neutrophil and T-cell XCIP results obtained, the 46 ET patients have been divided into three groups (Table 3).
Group A.
This group contains 23 patients (50%) in whom clonality analyses were
uninterpretable, either because of constitutive skewing of the
hematopoietic cells (ie, >75% expression of 1 allele), or because,
although the neutrophils and not the T cells were skewed, the patients
were over 65 years of age at the time of XCIP analysis. Seventeen
patients (1 through 17, Table 3) had a skewed XCIP in both neutrophils
and T cells and less than 20% difference between the two values for
each patient (Fig 2A). Six patients (18 through 23, Table 3) had apparent monoclonal neutrophils, but were less
than 65 years of age. Platelet RNA from 12 patients in this group was
also analyzed, and the XCIPs were the same as the neutrophil XCIPs, ie,
skewed, with less than 20% difference between the neutrophil and
platelet values for each patient. Altogether, 16 of the patients in
this group were greater than 65 years of age when XCIP analysis was
performed.
Group B. This group of 10 patients (24 through 33, Table 3), all less than 65 years of age, had monoclonal myelopoiesis with a skewed neutrophil XCIP, a balanced T-cell XCIP, and greater than 20% difference between the two results for each patient (Fig 2B). This represents 22% of the total patients studied and 43% of those with interpretable results. DNA analysis was performed on all patients and was confirmed with RNA analysis in 3 patients. Eight of these 10 patients had an extremely skewed myeloid XCIP with greater than 90% expression of one allele. Platelet RNA was analyzed in 3 patients, and in each case, the XCIP was again skewed in agreement with the neutrophil results. Group C.
The neutrophil and T-cell XCIPs of 12 patients (34 through 45, Table 3)
were both balanced and not significantly different from each other,
indicative of polyclonal myelopoiesis (Fig 2C). Results from all these
patients were obtained using DNA analysis and were confirmed by RNA
analysis in six patients. In patient no. 46 (Table 3), the neutrophil
and T-cell XCIPs had similar values, whether determined by DNA or RNA.
The DNA results were marginally skewed with neutrophil and T-cell
values of 81%:19% and 79%:21%, respectively, but the patient has
been assigned to group C (rather than A), because the values for the
RNA analysis were clearly balanced at 59%:41% and 66%:34%,
respectively. These 13 patients represent 28% of the total and 56% of
the evaluable patients. Five of these patients were older than 65 years
of age at the time of XCIP analysis. Platelet RNA was studied in 7 patients, and XCIPs were found to be balanced in agreement with the
neutrophil RNA data
XCIP Analysis of Myeloid Progenitors in ET Patients Myeloid progenitors were purified from bone marrow of 3 ET patients and XCIPs assessed using HUMARA analysis. In 1 patient with a balanced T-cell pattern and skewed neutrophil pattern (no. 24), the XCIPs from bone marrow cells, including the CD34+CD33
and CD34+CD33+ fractions, were also skewed (Fig
3 and Table 4). In 2 patients with balanced neutrophil and T-cell XCIPs
(no. 35 and 42), the CD34+CD33 and
CD34+CD33+ subpopulations also had balanced
XCIPs.
Clinical Comparison of Evaluable Patients With Clonal and Polyclonal Hematopoiesis The clinical records of patients with monoclonal (group B, patients no. 24 through 33, Tables 1 and 3) and polyclonal disease (group C, patients no. 34 through 46) were compared to determine whether there were any differences. Parameters examined were age and platelet count at diagnosis, length of follow-up, evidence of hepatomegaly or splenomegaly, and occurrence of thrombotic or hemorrhagic complications (Table 5). Statistical analysis showed no difference in age, platelet count, length of follow-up (Mann-U-Whitney), incidence of hepatosplenomegaly, or hemorrhagic complications (Fisher's exact test). However, thrombotic complications were more common in the monoclonal patients, and this difference was statistically significant (P = .039, Fisher's exact test). Thrombotic events were documented in 6 of the 10 monoclonal patients, and comprised 1 cerebrovascular accident, 3 transient ischemic episodes, 2 incidences of erythromelalgia, 2 deep venous thromboses, 1 pulmonary embolus, and 1 splenic infarct. Two of these patients had more than one event, and of these, one had multiple events, even when she had a normal platelet count. In the remaining 4 patients, the thrombotic episodes occurred at presentation. Two of the 13 polyclonal patients also had thromboses preceding the diagnosis of ET; 1 had recurrent miscarriages caused by placental infarction, and the other had a deep venous thrombosis after surgery for peripheral vascular disease. Interestingly, both of these patients had additional risk factors for thrombosis. The patient who had recurrent miscarriages was heterozygous for the factor V Leiden mutation and had anticardiolipin antibodies. The deep venous thrombosis of the other patient occurred immediately postoperatively.
This study illustrates the value of using DNA and RNA XCIPs to determine clonality in hematological disorders, but also shows some of the shortcomings of this technology. The results from only half of the 46 female patients were considered evaluable, and, because X chromosome inactivation only occurs in females, the diagnostic potential of XCIP analysis is thus limited to approximately one quarter of all patients with ET. Non-evaluability arises from either constitutive skewing of the hematopoietic cells, which is shown by analysis of the T cells, or from advanced patient age when acquired skewing in the myeloid cells may occur. We have chosen an arbitrary age cut-off, based on our previous studies11 of patients less than 65 years of age, to be able to diagnose clonality/oligoclonality of the myeloid cells. This does not mean that all analyses in patients greater than 65 years of age are unrewarding, because a polyclonal result is still interpretable.
Submitted June 3, 1998;
accepted September 21, 1998.
Address reprint requests to David C. Linch, FRCP, FRCPath, Department of Haematology, UCLMS, 98, Chenies Mews, London WC1E 6HX, UK.
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  E. Antonioli, P. Guglielmelli, G. Poli, C. Bogani, A. Pancrazzi, G. Longo, V. Ponziani, L. Tozzi, L. Pieri, V. Santini, et al. Influence of JAK2V617F allele burden on phenotype in essential thrombocythemia Haematologica, January 1, 2008; 93(1): 41 - 48. [Abstract] [Full Text] [PDF]
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 R. E. Gale, A. J.R. Allen, M. J. Nash, and D. C. Linch Long-term serial analysis of X-chromosome inactivation patterns and JAK2 V617F mutant levels in patients with essential thrombocythemia show that minor mutant-positive clones can remain stable for many years Blood, February 1, 2007; 109(3): 1241 - 1243. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
this includes patient no. 46, justifying their
inclusion in group C. In another patient who was not
informative for an RNA polymorphism (no. 42, Table 
, but that only neutrophils lacking the 20q
DNA probe.
Blood
83:931, 1994
-untranslated region of the thrombopoietin gene.
Blood
92:1091, 1998