Concomitant neutrophil JAK2V617F mutation screening and PRV-1 expression analysis in myeloproliferative disorders and secondary polycythaemia

Article (PDF Available)inBritish Journal of Haematology 131(2):166-71 · November 2005with26 Reads
DOI: 10.1111/j.1365-2141.2005.05743.x · Source: PubMed
Abstract
Polycythaemia vera (PV) is closely associated with both an acquired activating mutation of the JAK2 tyrosine kinase (JAK2(V617F)) in granulocyte-derived DNA and increased granulocyte polycythaemia rubra vera-1 (PRV-1) expression. In order to explore the correlation between these two biological markers and compare their diagnostic utility, mutation analysis for JAK2(V617F) and quantitative measurement of granulocyte PRV-1 expression were performed on the same study sample from 100 participants: 38 with PV, 22 with essential thrombocythaemia (ET), 10 with agnogenic myeloid metaplasia (AMM), 19 with secondary polycythaemia (SP) and 11 healthy volunteers. The respective overall (homozygous) JAK2(V617F) mutational frequencies were 95% (26%), 55% (0%), 30% (0%), 0% and 0%. The corresponding figures for increased PRV-1 expression were 89%, 18%, 20%, 21% and 9%. In patients with either ET or AMM, the likelihood of detecting JAK2(V617F) was significantly higher in the presence of an increased PRV-1 expression (83% vs. 38%; P = 0.05). Similarly, in patients with PV, homozygous as compared with heterozygous JAK2(V617F) correlated with higher levels of PRV-1 expression (P = 0.11). The present study suggests an allele dose-dependent effect of JAK2(V617F) on granulocyte PRV-1 expression. However, compared with the PRV-1 assay, mutation screening for JAK2(V617F) displayed greater accuracy in distinguishing PV from SP.
Concomitant neutrophil JAK2
V617F
mutation screening and PRV-
1 expression analysis in myeloproliferative disorders and
secondary polycythaemia
The majority (65–97%) of patients with polycythaemia vera
(PV) carry an acquired activating mutation of the JAK2
tyrosine kinase (JAK2
V617F
) in DNA derived from granulocytes
(Baxter et al, 2005; James et al, 2005; Jones et al, 2005;
Kralovics et al, 2005; Levine et al, 2005; Zhao et al, 2005).
The same mutation is also found frequently in other BCR/
ABL-negative myeloproliferative disorders (MPD) including
essential thrombocythaemia (32–57%) and agnogenic myeloid
metaplasia (AMM) (43–50%) (Baxter et al, 2005; James et al,
2005; Jones et al, 2005; Kralovics et al, 2005; Levine et al,
2005) and less frequently in chronic myelomonocytic leukae-
mia, myelodysplastic syndrome, systemic mastocytosis, chro-
nic neutrophilic leukaemia, eosinophilic disorders and atypical
MPD (Jelinek et al, 2005; Jones et al, 2005; Steensma et al,
2005). However, to date, JAK2
V617F
has not been reported in
either normal controls (Baxter et al, 2005; James et al, 2005;
Kralovics et al, 2005; Levine et al, 2005) or patients with
secondary erythrocytosis (James et al, 2005; Kralovics et al,
2005). Therefore, detection of the JAK2
V617F
mutant allele
might carry a strong positive-predictive value in distinguishing
MPD, including PV, from non-clonal conditions, such as
secondary polycythaemia (SP).
Previous studies have also demonstrated a close association
between PV and increased granulocyte polycythaemia rubra
vera-1 (PRV-1) expression that was neither invariable (test
sensitivity ranged from 69% to 100%) nor exclusive
(a substantial minority of patients with essential thrombo-
cythaemia (ET), AMM and other myeloid disorders also
display the specific abnormality) (Temerinac et al , 2000;
Kralovics et al, 2003; Liu et al, 2003; Florensa et al, 2004;
Ayalew Tefferi,
1
Shireen Sirhan,
1
Terra L.
Lasho,
1
Susan M. Schwager,
1
Chin-Yang
Li,
1
David Dingli,
1
Alexandra P.
Wolanskyj,
1
David P. Steensma,
1
Ruben
Mesa
1
and D. G. Gilliland
2
1
Mayo Clinic and Mayo Clinic College of
Medicine, Rochester, MN, and
2
Howard Hughes
Institute and Harvard Medical School, Boston,
MA, USA
Received 20 June 2005; accepted for publication
29 July 2005
Correspondence: Ayalew Tefferi, MD, Mayo
Clinic and Mayo Clinic College of Medicine, 200
First Street SW, Rochester, MN 55905, USA.
E-mail: tefferi.ayalew@mayo.edu
Summary
Polycythaemia vera (PV) is closely associated with both an acquired
activating mutation of the JAK2 tyrosine kinase (JAK2
V617F
)in
granulocyte-derived DNA and increased granulocyte polycythaemia rubra
vera-1 (PRV-1) expression. In order to explore the correlation between these
two biological markers and compare their diagnostic utility, mutation
analysis for JAK2
V617F
and quantitative measurement of granulocyte PRV-1
expression were performed on the same study sample from 100 participants:
38 with PV, 22 with essential thrombocythaemia (ET), 10 with agnogenic
myeloid metaplasia (AMM), 19 with secondary polycythaemia (SP) and 11
healthy volunteers. The respective overall (homozygous) JAK2
V617F
mutational frequencies were 95% (26%), 55% (0%), 30% (0%), 0% and
0%. The corresponding figures for increased PRV-1 expression were 89%,
18%, 20%, 21% and 9%. In patients with either ET or AMM, the likelihood
of detecting JAK2
V617F
was significantly higher in the presence of an increased
PRV-1 expression (83% vs. 38%; P ¼ 0Æ05). Similarly, in patients with PV,
homozygous as compared with heterozygous JAK2
V617F
correlated with
higher levels of PRV-1 expression (P ¼ 0Æ 11). The present study suggests an
allele dose-dependent effect of JAK2
V617F
on granulocyte PRV-1 expression.
However, compared with the PRV-1 assay, mutation screening for JAK2
V617F
displayed greater accuracy in distinguishing PV from SP.
Keywords: polycythaemia, myeloproliferative, secondary, mutation, gene
expression, diagnosis.
research paper
doi:10.1111/j.1365-2141.2005.05743.x ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 131, 166–171
Koch et al, 2004; Passamonti et al, 2004; Tefferi et al, 2004;
Sirhan et al, 2005). In contrast to that observed with mutation
analysis for JAK2
V617F
, increased PRV-1 expression has been
reported in both normal controls and patients with SP
(Passamonti et al, 2004; Sirhan et al, 2005). On the contrary,
both JAK2
V617F
and increased granulocyte PRV-1 expression
display a significantly higher prevalence in PV and post-
polycythaemic myeloid metaplasia as opposed to other MPD,
suggesting a correlation between the two markers (Tefferi et al,
2004; Baxter et al, 2005; James et al, 2005; Kralovics et al,
2005; Levine et al, 2005). The present study explored this
possibility and compared the diagnostic utility of the two
laboratory markers in distinguishing PV from SP.
Methods
The current study was approved by the Mayo Clinic
institutional review board (IRB) and involved 100 consecu-
tive patients in whom peripheral blood samples were
collected between April 2003 and July 2004 under an IRB-
approved protocol. The diagnoses of PV, ET and AMM were
made according to the World Health Organisation (WHO)
diagnostic criteria (Jaffe et al, 2001). The designation of SP
required the presence of a comorbidity known to be
associated with SP as well as a bone marrow examination
that was felt to be inconsistent with a primary myeloid
disorder. Bone marrow histology in all cases was reviewed by
Mayo Clinic haematopathologists and re-reviewed by one of
the authors.
Both JAK2 mutational analysis and measurement of PRV-1
expression were performed on the granulocyte fraction of
peripheral blood. Double density gradient centrifugation (Hist-
opaque-1077
TM
layered over Histopaque-1119
TM
; Sigma Diag-
nostics, St Louis, MO, USA) was used to separate out the
granulocyte cell layer from each sample. Genomic DNA was
extracted using QIAamp Blood Mini Kit (Qiagen, Valencia, CA,
USA) and amplified by polymerase chain reaction (PCR). Each
50-ll PCR reaction contained approximately 25 ng of DNA
template, 5 ll 10X Roche Buffer (final concentration of MgCl
2
:
1Æ5 mmol/l
)1
), 1Æ5UTaq polymerase (Roche, Indianapolis, IN,
USA), 0Æ8 mmol/l
)1
dNTPs (Roche), and 20 pmol/l
)1
each of
sense and antisense primers (5¢-TGCTGAAAGTAGGAGA
AAGTGCAT-3¢ and 5¢-TCCTACAGTGTTTTCAGTTTCAA-3¢
respectively).
The PCR cycling parameters were: one cycle of 94C for
2 min; 35 cycles of 94C for 30 s, 52C for 40 s and 72C for
40 s; followed by one cycle of 72C for 2 min. PCR products
were cleaned with QIAquick PCR purification Kit (Qiagen).
Fluorescent dye chemistry sequencing was performed using the
same primers used for amplification, on an ABI Prism 3700
DNA Analyzer (Applied Biosystems, Foster City, CA, USA).
Sequencher 4.2 (Gene Codes Corporation, Ann Arbor, MI,
USA) and GenBank accession NM_004972 (JAK2 mRNA) and
the corresponding region from the NC_000009 chromosome 9
contig were used for sequence analysis.
The PRV-1 mRNA level was quantified by real-time reverse
transcription-PCR according to the previously published
methods (Klippel et al, 2003). To standardise results, the
experiment was run as a relative quantification assay, incor-
porating the housekeeping glyceraldehyde-3-phosphate dehy-
drogenase (GAPDH) gene transcript. The mean cycle threshold
(C
T
) value for PRV-1 was divided by the mean C
T
value for
GAPDH, creating a PRV-1/GAPDH ratio. Consequently, a low
PRV-1/GAPDH C
T
ratio indicated increased PRV-1 mRNA
level. PRV-1 gene expression was considered elevated in the
presence of a PRV-1/GAPDH ratio of 1Æ17 or lower (Klippel
et al, 2003).
Statistical comparison between categorical variables was
performed by chi-squared statistics. Comparison between
categorical and continuous variables was performed by either
the Mann–Whitney U-test or Kruskal–Wallis test. All data
were analysed by using sas software (SAS, Inc., Cary, NC,
USA).P <0Æ05 were considered statistically significant.
Results
Among the 100 study patients, 38 had PV, 22 had ET, 10 had
AMM, 19 had SP and 11 were healthy volunteers. Comorbi-
dities associated with SP included obstructive sleep apnoea
with or without additional lung disease or tobacco use in five
patients, treatment with testosterone in four patients, heavy
tobacco use with or without associated lung disease in three
patients, haemoglobinopathies with increased oxygen affinity
in two patients, chronic obstructive pulmonary disease in two
patients, and one patient each with atrial septal defect and
pulmonary stenosis, renal cyst and high altitude habitat.
Baseline patient characteristics and the results of both
JAK2
V617F
mutation analysis and real-time PCR measurement
of granulocyte PRV-1 transcript levels are presented in Table I.
The age distributions were similar among the different disease
categories but there were more males represented in the AMM
and SP disease category. However, currently there is no
evidence with regard to gender bias in terms of either the
presence of the JAK2
V617F
mutation in AMM or neutrophil
PRV-1 expression in SP. The same can be said regarding
disease duration in general although two studies have sugges-
ted a longer disease duration associated with homozygous
JAK2
V617F
in MPD (Kralovics et al, 2005; Levine et al, 2005).
As expected, increased neutrophil PRV-1 expression had the
highest frequency in PV (89%) and was less prevalent in the
other MPD including ET (18%) and AMM (20%). More
importantly, abnormal levels were also registered in 21% of
patients with SP and one of 11 (9%) healthy volunteers. In
contrast, the JAK2
V617F
mutation was not detected in the
absence of clonal myelopoiesis (a test specificity of 100%). In
the current study, we found the mutational frequency in PV to
be over 90% providing a test sensitivity that was comparable
with that of neutrophil PRV-1 expression. However, both
biological markers were suboptimal in distinguishing PV from
other MPD (Table I).
PRV-1 and JAK2
V617F
in MPD
ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 131, 166–171 167
There was a significant across the board correlation between
the presence of JAK2
V617F
mutation in MPD and neutrophil
PRV-1 expression (Fig 1, P ¼ 0Æ0001). When the analysis was
restricted to patients with either ET or AMM, patients with the
mutation (n ¼ 15) compared with those without (n ¼ 17)
were significantly more likely to display increased PRV-1
expression (P ¼ 0Æ001). Similarly, the 10 PV patients who were
homozygous for the mutant allele had a higher PRV-1
transcript level compared with the 26 PV patients who were
heterozygous for the mutation, although the difference did not
reach statistical significance (P ¼ 0Æ11).
Discussion
The results of the current study confirm a high positive-
predictive value (100%) for MPD in the presence of JAK2
V617F
and support the incorporation of mutation screening for
JAK2
V617F
during the evaluation of polycythaemia as well as
thrombocythaemia (Fig 2 and 3). In this regard, its value
might supersede that of the PRV-1 assay and other specialised
tests for distinguishing PV from SP including platelet-rich
plasma serotonin level (Koch et al, 2004), endogenous in vitro
erythroid colony formation (Reid, 1987), and megakaryocyte/
platelet Mpl expression (Moliterno et al, 1998; Tefferi et al,
2000), each of which have important limitations. Furthermore,
diagnostic accuracy might be enhanced by the use of more
sensitive techniques that detect JAK2
V617F
in minor clonal cell
populations, such as allele-specific PCR (Baxter et al, 2005;
Jones et al, 2005). However, it is underscored that mutation
screening for JAK2
V617F
is not helpful in distinguishing the
different categories of chronic myeloid disorders. For com-
parative purposes, we have included the current WHO criteria
for the diagnosis of PV (Table II) and ET (Table III).
The present study also demonstrated an allele dose-depend-
ent significant association between the presence of JAK2
V617F
and increased PRV-1 expression (Fig 1). In patients with either
ET or AMM, for example, five of the six patients (83%) with
increased PRV-1 expression also carried the JAK2
V617F
mutant
allele as opposed to only 10 of the 26 (38%) with normal
PRV-1 expression (P ¼ 0Æ05). Similarly, PV patients with
homozygous JAK2
V617F
, compared with PV patients having
Table I. Baseline patient characteristics and
summary of JAK2
V617F
mutation analysis as well
as quantitative measurement of granulocyte
PRV-1 expression in 100 patients with poly-
cythaemia vera (PV), essential thrombocythae-
mia (ET), agnogenic myeloid metaplasia
(AMM), secondary polycythaemia (SP) or
healthy volunteers.
PV
(n ¼ 38)
ET
(n ¼ 22)
AMM
(n ¼ 10)
SP
(n ¼ 19)
Controls
(n ¼ 11)
Age (years)
Median 58 46 57 57 N/A
Range 18–81 18–81 41–67 23–77
Sex (f/m) 20/18 14/8 1/9 3/16 N/A
Disease duration (months)
Median 34 22Æ5 9 2 N/A
Range 0–290 0–152 0–348 0–120
PRV-1/GAPDH
Median 1Æ01 1Æ23 1Æ31 1Æ25 1Æ25
Range 0Æ83–1Æ28 0Æ9–1Æ44 1Æ08–1Æ43 1Æ14–1Æ39 1Æ14–1Æ43
% with increased value* 89 18 20 21 9
JAK2
V617
Absent (%) 2 (5) 10 (45) 7 (70) 20 (100) 11 (100)
Present (%) 36 (95) 12 (55) 3 (30) 0 0
Homozygous (%) 10 (26) 0 0
PRV-1, polycythaemia rubra vera-1; GADPH, glyceraldehyde-3-phosphate dehydrogenase; N/A,
not applicable.
*Neutrophil PRV-1 expression was considered increased when the neutrophil PRV-1/GAPDH
ratio was below 1Æ17 (Klippel et al, 2003).
0·9
0·8
1
·11
·12
·13
·14
·15
PRV-1/GAPDH
i
W
d
l-
ty
pe H
e
t
e
zory suogH
om
oz
y
suog
P
< 0·0001
Fig 1. Distribution of granulocyte polycythaemia rubra vera-1 (PRV-
1) and glyceraldehyde-3-phosphate dehydrogenase ratio in 70 patients
with myeloproliferative disorders including 38 with polycythaemia
vera, 22 with essential thrombocythemia and 10 with agnogenic my-
eloid metaplasia. The figure displays a significantly altered PRV-1
expression based on mutation status for JAK2; wild-type versus het-
erozygous JAK2
V617F
versus homozygous JAK2
V617F
. Boxes enclose
values within the first and third quartile ranges divided by a line
representing the median. Whiskers indicate 1Æ5 · the interquartile
range above and below the 75th and 25th percentile.
A. Tefferi et al
168
ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 131, 166–171
heterozygous mutations, displayed higher levels of granulocyte
PRV-1 expression (P ¼ 0Æ11). Taken together, the particular
observation suggests a JAK2-mediated effect on granulocyte
PRV-1 expression and is consistent with previously reported
observations that have linked JAK2
V617F
with the PV pheno-
type including the induction of erythrocytosis in mice after
transplanting them with murine bone marrow cells transduced
with murine JAK2
V617F
(James et al, 2005), the demonstration
Serum erythropoietin level
Low Normal Increased
Presence of PV-characteristic
clinical or laboratory features*
Ye s No
PV diagnosis
unlikely
Bone marrow biopsy
and mutation screening
for JAK2
Two options
Recheck CBC
periodically
Peripheral blood JAK2
mutation screening
PV diagnosis likely in
the presence of either a
JAK2 mutation or
consistent histology
A JAK2 mutation does not
distinguish PV from other
MPD
Bone marrow biopsy if
positive, otherwise recheck
CBCB periodically
Fig 2. A diagnostic algorithm for polycythaemia vera (PV) that incorporates mutation screening for JAK2
V617F
. PV-characteristic features include
increased leucocyte alkaline phosphatase score, thrombocytosis, leucocytosis, splenomegaly, thrombosis, pruritus and erythromelalgia. MPD, my-
eloproliferative disorder; CBC, complete blood count. *Please note that the current algorithm has not been validated by a systematic study and is
based on a consensus working procedure that is currently in use at the authors’ institutions. Furthermore, the algorithm assumes the use of a properly
validated modern assay for serum erythropoietin determination (Ma et al, 1992; Mossuz et al, 2004).
Bone marrowbiopsy, cytogeneticstudies,FISHor
RT-PCR for BCR/ABL,andmutationscreening for JAK2
BCR/ABL -positive
JAK2-mutation
present
No
Chronic myeloid
leukemia
Either other cytogenetic
abnormalities present or
histology consistent with
MPD
Yes No
Use bone marrow
histology to distinguish
ET from other MPD
ET unlikely
Use bone marrow
histology to
distinguish ET
from other MPD
MPD
Fig 3. A diagnostic algorithm for primary thrombocythaemia (i.e. clinically not consistent with reactive thrombocytosis). ET, essential thrombo-
cythaemia; MPD, myeloproliferative disorder; FISH, fluorescence in situ hybridisation; RT-PCR, reverse transcription-polymerase chain reaction.
*Please note that the current algorithm has not been validated by a systematic study and is based on a consensus working procedure that is currently
in use at the authors’ institutions.
PRV-1 and JAK2
V617F
in MPD
ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 131, 166–171 169
of JAK2
V617F
-mediated cytokine hypersensitivity in cell lines
(James et al, 2005; Kralovics et al, 2005; Levine et al, 2005), the
invariable presence of JAK2
V617F
in epo-independent erythroid
colonies (Baxter et al, 2005), and the association in ET of the
mutant allele with higher haemoglobin levels at diagnosis
(Wolanskyj et al, 2005).
We entertained two possibilities, without the exclusion of
others, to explain the significant association between homo-
zygous JAK2
V617F
and PRV-1 expression. In a recent commu-
nication, serial analysis of archived bone marrow cells in
JAK2
V617F
homozygote patients disclosed a gradual change
from a heterozygous to homozygous mutational status over
time that suggested clonal dominance rather than a two-step
molecular event (Tefferi et al, 2005). As such, approximately
half of the neutrophils in JAK2
V617F
‘heterozygotes’ might not
be clonally involved and thus do not contribute to the PRV-1
signal. The second explanation recalls previous observations
regarding increased PRV-1 expression in several reactive
conditions as well as in growth factor-stimulated granulocy-
tosis that raised the possibility of the phenomenon being a
marker of neutrophil activation (Passamonti et al, 2004).
Accordingly, the JAK2
V617F
-associated increase in PRV-1
might represent a recapitulation of cytokine-associated neu-
trophil activation that is known to be mediated through the
JAK-signal transducer and activator of transcription signalling
pathway (Al-Shami et al, 1998). Finally, two recent commu-
nications have also demonstrated the close association between
PRV-1 expression and JAK2
V617F
in MPDs (Goerttler et al,
2005; Jones et al, 2005).
References
Al-Shami, A., Mahanna, W. & Naccache, P.H. (1998) Granulocyte-
macrophage colony-stimulating factor-activated signaling pathways
in human neutrophils. Selective activation of Jak2, Stat3, and Stat5b.
Journal of Biological Chemistry, 273, 1058–1063.
Baxter, E.J., Scott, L.M., Campbell, P.J., East, C., Fourouclas, N.,
Swanton, S., Vassiliou, G.S., Bench, A.J., Boyd, E.M., Curtin, N.,
Scott, M.A., Erber, W.N. & Green, A.R. (2005) Acquired mutation of
the tyrosine kinase JAK2 in human myeloproliferative disorders.
Lancet, 365, 1054–1061.
Florensa, L., Besses, C., Zamora, L., Bellosillo, B., Espinet, B., Serrano,
S., Woessner, S. & Sole, F. (2004) Endogenous erythroid and
megakaryocytic circulating progenitors, HUMARA clonality assay,
and PRV-1 expression are useful tools for diagnosis of polycythemia
vera and essential thrombocythemia. Blood, 103, 2427–2428.
Goerttler, P.S., Steimle, C., Marz, E., Johansson, P.L., Andreasson, B.,
Griesshammer, M., Gisslinger, H., Heimpel, H. & Pahl, H.L. (2005)
The Jak2V617F mutation, PRV-1 overexpression and EEC forma-
tion define a similar cohort of MPD patients. Blood, doi: 10.1182/
blood-2005–04–1515. Prepublished online 28 June 2005.
Jaffe, E.S., Harris, N.L., Stein, H. & Vardiman, J.W. (eds) (2001) WHO
Classification of Tumors: Tumors of the Hematopoietic and Lymphoid
Tissues. IARC Press, Lyon.
James, C., Ugo, V., Le Couedic, J.P., Staerk, J., Delhommeau, F.,
Lacout, C., Garcon, L., Raslova, H., Berger, R., Bennaceur-Griscelli,
Table III. World Health Organisation (WHO) criteria for essential
thrombocythaemia (ET), Jaffe et al (2001).
Positive criteria
1. Sustained platelet count 600 · 10
9
/l
2. Bone marrow biopsy specimen showing proliferation mainly
of the megakaryocytic lineage with increased numbers of enlarged,
mature megakaryocytes
Criteria of exclusion
1. No evidence of polycythaemia vera (PV)
a. Normal red cell mass or hemoglobin <18Æ5 g/dl in
men, 16Æ5 g/dl in women
b. Stainable iron in marrow, normal serum ferritin
or normal mean cell volume (MCV)
c. If the former condition is not met, failure of iron
trial to increase red cell mass or hemoglobin levels to the PV range
2. No evidence of chronic myeloid leukaemia
a. No Philadelphia chromosome and no BCR/ABL fusion gene
3. No evidence of chronic idiopathic myelofibrosis
a. Collagen fibrosis absent
b. Reticulin fibrosis minimal or absent
4. No evidence of myelodysplastic syndrome
a. No del(5q), t(3;3)(q21;q26), inv(3)(q21q26)
b. No significant granulocytic dysplasia,
few if any micromegakaryocytes
5. No evidence that thrombocytosis is reactive because of
a. Underlying inflammation or infection
b. Underlying neoplasm
c. Prior splenectomy
Table II. World Health Organisation (WHO) criteria for polycythae-
mia vera (PV).
A-criteria
1. Elevated red cell mass >25% above mean normal predicted value,
or hemoglobin >18Æ5 g/dl in men, 16Æ5 g/dl in women or >99th
percentile of method-specific reference range for age, sex,
altitude of residence.
2. No cause of secondary erythrocytosis, including
a. Absence of familial erythrocytosis
b. No elevation of erythropoietin because of;
i. Hypoxia (arterial pO
2
£ 92%)
ii. High oxygen affinity haemoglobin
iii. Truncated erythropoietin receptor
iv. Inappropriate erythropoietin production by tumour
3. Splenomegaly
4. Clonal genetic abnormality other than Philadelphia
chromosome or BCR/ABL fusion gene in marrow cells
5. Endogenous erythroid colony formation in vitro
B-criteria
1. Thrombocytosis >400 · 10
9
/l
2. Leucocytosis >12 · 10
9
/l
3. Bone marrow biopsy showing panmyelosis with
prominent erythroid and megakaryocytic proliferation
4. Low serum erythropoietin levels
Diagnosis of PV requires the presence of the first two A criteria
together with either any one other A criterion or two B criteria (Jaffe
et al, 2001).
A. Tefferi et al
170
ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 131, 166–171
A., Villeval, J.L., Constantinescu, S.N., Casadevall, N. & Vainchen-
ker, W. (2005) A unique clonal JAK2 mutation leading to consti-
tutive signalling causes polycythemia vera. Nature 434, 1144–1148.
Jelinek, J., Oki, Y., Gharibyan, V., Bueso-Ramos, C., Prchal, J.T.,
Verstovsek, S., Beran, M., Estey, E., Kantarjian, H.M. & Issa, J.P.
(2005) JAK2 mutation 1849G >T is rare in acute leukemias but can
be found in CMML, Philadelphia-chromosome negative CML and
megakaryocytic leukemia. Blood, doi: 10.1182/blood-2005–05–1800.
Prepublished online 21 July 2005.
Jones, A.V., Kreil, S., Zoi, K., Waghorn, K., Curtis, C., Zhang, L., Score,
J., Seear, R., Chase, A.J., Grand, F.H., White, H., Zoi, C., Louko-
poulos, D., Terpos, E., Vervessou, E.C., Schultheis, B., Emig, M.,
Ernst, T., Lengfelder, E., Hehlman, R., Hochhaus, A., Oscier, D.,
Silver, R.T., Reiter, A. & Cross, N.C.P. (2005) Widespread occur-
rence of the JAK2 V617F mutation in chronic myeloproliferative
disorders. Blood, doi: 10Æ1182/blood-2005–03–1320. Prepublished
online 26 May 2005.
Klippel, S., Strunck, E., Temerinac, S., Bench, A.J., Meinhardt, G.,
Mohr, U., Leichtel, R., Green, A.R., Greisshammer, M., Heimpel, H.
& Pahl, H.L. (2003) Quantification of PRV-1 mRNA distinguishes
polycythemia vera from secondary erythrocytosis. Blood, 102, 3569–
3574.
Koch, C.A., Lasho, T.L. & Tefferi, A. (2004) Platelet-rich plasma ser-
otonin levels in chronic myeloproliferative disorders: evaluation of
diagnostic use and comparison with the neutrophil PRV-1 assay.
British Journal of Haematology, 127, 34–39.
Kralovics, R., Buser, A.S., Teo, S.S., Coers, J., Tichelli, A., van der Maas,
A.P. & Skoda, R.C. (2003) Comparison of molecular markers in a
cohort of patients with chronic myeloproliferative disorders. Blood,
102, 1869–1871.
Kralovics, R., Passamonti, F., Buser, A.S., Soon-Siong, T., Tiedt, R.,
Passweg, J.R., Tichelli, A., Cazzola, M. & Skoda, R.C. (2005) A gain
of function mutation in Jak2 is frequently found in patients with
myeloproliferative disorders. New England Journal of Medicine, 352,
1779–1790.
Levine, R.L., Wadleigh, M., Cools, J., Ebert, B.L., Wernig, G., Huntly,
B.J., Boggon, T.J., Wlodarska, I., Clark, J.J., Moore, S., Adelsperger,
J., Koo, S., Lee, J.C., Gabriel, S., Mercher, T., D’Andrea, A., Froh-
ling, S., Dohner, K., Marynen, P., Vandenberghe, P., Mesa, R.A.,
Tefferi, A., Griffin, J.D., Eck, M.J., Sellers, W.R., Meyerson, M.,
Golub, T.R., Lee, S.J. & Gilliland, D.G. (2005) Activating mutation
in the tyrosine kinase JAK2 in polycythemia vera, essential throm-
bocythemia, and myeloid metaplasia with myelofibrosis. Cancer
Cell, 7, 387–397.
Liu, E., Jelinek, J., Pastore, Y.D., Guan, Y., Prchal, J.F. & Prchal, J.T.
(2003) Discrimination of polycythemias and thrombocytoses by
novel, simple, accurate clonality assays and comparison with PRV-1
expression and BFU-E response to erythropoietin. Blood, 101, 3294–
3301.
Ma, D.D., Wei, A.Q., Dowton, L.A., Lau, K.S., Wu, Z.H. & Ueda, M.
(1992) Assessment of an EIA for measuring human serum ery-
thropoietin as compared with RIA and an in-vitro bioassay. British
Journal of Haematology, 80, 431–436.
Moliterno, A.R., Hankins, W.D. & Spivak, J.L. (1998) Impaired
expression of the thrombopoietin receptor by platelets from patients
with polycythemia vera. New England Journal of Medicine, 338, 572–
580.
Mossuz, P., Girodon, F., Donnard, M., Latger-Cannard, V., Dobo, I.,
Boiret, N., Lecron, J.C., Binquet, C., Barro, C., Hermouet, S. &
Praloran, V. (2004) Diagnostic value of serum erythropoietin level in
patients with absolute erythrocytosis. Haematologica, 89, 1194–1198.
Passamonti, F., Pietra, D., Malabarba, L., Rumi, E., Della Porta, M.G.,
Malcovati, L., Bonfichi, M., Pascutto, C., Lazzarino, M. & Cazzola,
M. (2004) Clinical significance of neutrophil CD177 mRNA ex-
pression in Ph-negative chronic myeloproliferative disorders. British
Journal of Haematology, 126, 650–656.
Reid, C.D. (1987) The significance of endogenous erythroid colonies
(EEC) in haematological disorders. Blood Reviews, 1, 133–140.
Sirhan, S., Lasho, T.L., Elliott, M.A. & Tefferi, A. (2005) Neutrophil
polycythemia rubra vera-1 expression in classic and atypical mye-
loproliferative disorders and laboratory correlates. Haematologica,
90, 406–408.
Steensma, D.P., Dewald, G.W., Lasho, T.L., Powell, H.L., McClure,
R.F., Levine, R.L., Gilliland, D.G. & Tefferi, A. (2005) The JAK2
V617F activating tyrosine kinase mutation is an infrequent event in
both ‘‘atypical’’ myeloproliferative disorders and the myelodysplas-
tic syndrome. Blood, 106, 1207–1209.
Tefferi, A., Yoon, S.Y. & Li, C.Y. (2000) Immunohistochemical staining
for megakaryocyte c-mpl may complement morphologic distinction
between polycythemia vera and secondary erythrocytosis. Blood, 96,
771–772.
Tefferi, A., Lasho, T.L., Wolanskyj, A.P. & Mesa, R.A. (2004) Neu-
trophil PRV-1 expression across the chronic myeloproliferative
disorders and in secondary or spurious polycythemia. Blood, 103,
3547–3548.
Tefferi, A., Lasho, T.L., Gilliland, D.G. & Dewald, G. (2005) Is
homozygosity for JAK2 V617F in myeloproliferative disorders a
time-acquired phenomenon?. New England Journal of Medicine (in
press).
Temerinac, S., Klippel, S., Strunck, E., Roder, S., Lubbert, M., Lange,
W., Azemar, M., Meinhardt, G., Schaefer, H.E. & Pahl, H.L. (2000)
Cloning of PRV-1, a novel member of the uPAR receptor super-
family, which is overexpressed in polycythemia rubra vera. Blood, 95,
2569–2576.
Wolanskyj, A.P., Lasho, T.L., Schwager, S.M., Mcclure, R.F., Wadleigh,
M., Lee, S.J., Gilliland, D.G. & Tefferi, A. (2005) JAK2
V617F
mutation in essential thrombocythaemia: clinical associations and
long-term prognostic relevance. British Journal of Haematology
(in press).
Zhao, R., Xing, S., Li, Z., Fu, X., Li, Q., Krantz, S.B. & Zhao, Z.J. (2005)
Identification of an acquired JAK2 mutation in polycythemia vera.
Journal of Biological Chemistry, 280, 22788–22792.
PRV-1 and JAK2
V617F
in MPD
ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 131, 166–171 171
    • "They also did not find the mutation when examining 11 healthy subjects without secondary polycythemia. Method sensitivity was not reported [34]. Tagariello et al. found the mutation in one blood donor after comparing 84 men and 19 women with elevated hematocrit (50 and 46%, respectively ) to 79 normal hematocrit controls using ARMS method with a 1–2% sensitivity. "
    [Show abstract] [Hide abstract] ABSTRACT: The JAK2 V617F mutation is responsible for the constitutive activation of the erythropoietin receptor signaling pathway in most cases of polycythemia vera (PV). The mutation has also been described in healthy people. As smoking may result in secondary polycythemia, the goal of this trial was to examine the effect of smoking on the prevalence of the JAK2 mutation and its correlation to erythrocytosis. The study was case-control. Hospitalized smokers (n = 81) and nonsmokers (n = 61) were recruited. Serum was drawn for complete blood count, erythropoietin, ferritin and venous blood gases. JAK2 mutation was analyzed by highly sensitive allele-specific Quantitative Real Time PCR. The JAK2 mutation was found in 29/81 (35.8%) of smokers in comparison to only 9/61 (14.8%) of the control group (P = 0.007). The frequency of the mutation among smokers who were positive for the JAK2 mutation had a mean of 6.78 × 10(-4) ± 1.08 × 10(-3) vs. 1.51 × 10(-4) ± 2.04 × 10(-4) among nonsmokers (P = 0.027). Both frequencies are much lower than those found in PV. There was a medium correlation between older age and mutation frequency in nonsmokers (r= 0.67, P = 0.043). Hematocrit was higher in smokers (47.8 ± 6 vs. 41.7 ± 4.7, P < 0.0001), but no correlation was found to JAK2 mutation. In a cohort of hospitalized smokers and nonsmokers, JAK2 mutation was more prevalent and found in higher frequencies among smokers than nonsmokers. We suggest that accelerated erythropoiesis renders the cells susceptible to JAK2 mutation.
    Full-text · Article · Jan 2012
    • "Subsequently, the mutation has also described in other myeloid neoplasms [103, 119]. As of the time of this writing, JAK2V617F has not been reported in lymphoid disorders120121122123, solid tumour124125126 or secondary myeloproliferation [127, 128] [142]. However, the interaction between JAK2 V617F homozygosity and mutant allele burden, in terms of phenotypic determination, is currently not clear [143, 144]. "
    [Show abstract] [Hide abstract] ABSTRACT: Therapeutically validated oncoproteins in myeloproliferative neoplasms (MPN) include BCR-ABL1 and rearranged PDGFR proteins. The latter are products of intra- (e.g. FIP1L1-PDGFRA) or inter-chromosomal (e.g. ETV6-PDGFRB) gene fusions. BCR-ABL1 is associated with chronic myelogenous leukaemia (CML) and mutant PDGFR with an MPN phenotype characterized by eosinophilia and in addition, in case of FIP1L1-PDGFRA, bone marrow mastocytosis. These genotype-phenotype associations have been effectively exploited in the development of highly accurate diagnostic assays and molecular targeted therapy. It is hoped that the same will happen in other MPN with specific genetic alterations: polycythemia vera (JAK2 V617F and other JAK2 mutations), essential thrombocythemia (JAK2V617F and MPL515 mutations), primary myelofibrosis (JAK2 V617F and MPL515 mutations), systemic mastocytosis (KITD816V and other KIT mutations) and stem cell leukaemia/lymphoma (ZNF198-FGFR1 and other FGFR1 fusion genes). The current review discusses the above listed mutant molecules in the context of their value as drug targets.
    Article · Nov 2008
    • "However, JAK2 V617F is rarely identified in healthy controls or patients with other myeloid disorders. Thus, JAK2 V617F has general diagnostic value for MPN, but it cannot be used to differentiate between PV, ET, or PMF [1, 2,45678910111213141516171819. The diagnostic utility of JAK2 V617F mutation screening in hypercellular bone marrow specimens with fibrosis has not been previously investigated. "
    [Show abstract] [Hide abstract] ABSTRACT: The myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS) occasionally demonstrate overlapping morphological features including hypercellularity, mild/nonspecific dysplastic changes and variable bone marrow fibrosis. Thus, when the associated bone marrow fibrosis results in a suboptimal specimen for morphological evaluation, the descriptive diagnosis "fibrotic marrow with features indeterminate for MDS versus MPN" is often applied. The JAK2 ( V617F ) mutation was recently shown to be frequently identified in MPN, but it is rarely present in other myeloid disorders. However, the diagnostic utility of JAK2 ( V617F ) screening in hypercellular bone marrow specimens with fibrosis has not been previously investigated. Using a real-time polymerase chain reaction melting-curve assay capable of detecting JAK2 ( V617F ) in archived fixed materials, we retrospectively studied JAK2 ( V617F ) in 45 cases with fibrotic hypercellular bone marrow at initial presentation, including 19 cases initially described as "with features indeterminate for MDS versus MPN". These 19 cases were reclassified into more specific categories of MDS (n = 14) or MPN (n = 5) based on the availability of subsequent clinical data and/or bone marrow examinations. The JAK2 ( V617F ) allele was identified in 17 out of 18 BCR/ABL gene-negative MPN cases with marrow fibrosis, whereas only wild-type alleles were identified in the remaining non-MPN cases. Importantly, JAK2 ( V617F ) alleles were seen in all five cases of "with features indeterminate for MDS versus MPN" at initial presentation that were later determined to be MPN, but they were absent in the 14 cases later determined to be MDS. Our results suggest that JAK2 ( V617F ) allele evaluation can be a useful ancillary test for discriminating MDS from MPN in specimens with bone marrow fibrosis.
    Full-text · Article · Oct 2008
Show more

Recommended publications

Discover more