The JAK2 V617F tyrosine kinase mutation in myelodysplastic syndromes (MDS) developing myelofibrosis indicates the myeloproliferative nature in a subset of MDS patients [14]

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The JAK2 V617F tyrosine kinase mutation in myelodysplastic syndromes (MDS)
developing myelofibrosis indicates the myeloproliferative nature in a subset
of MDS patients
Leukemia (2005) 19, 2359–2360. doi:10.1038/sj.leu.2403989;
published online 20 October 2005
TO THE EDITOR
The JAK2 V617F tyrosine kinase mutation is responsible for the
development of BCR/ABL-negative chronic myeloproliferative
disorders, including myelofibrosis (MF).
1–4
Kralovics et al
1
demonstrated that 74% of patients with polycythemia vera
(PV), 32% of essential thrombocythemia (ET), and 35% of
patients with primary MF harbored the JAK2 V617F mutation.
Recently, Steensma et al
5
demonstrated that 5% (five of 101
patients) of myelodysplastic syndromes (MDS) patients had the
JAK2 V617F tyrosine kinase mutation and reported its infrequent
occurrence in other myeloid disorders, including MDS. How-
ever, the exact significance of the JAK2 V617F mutation in MDS
is obscure. Myelofibrosis is associated with various hematologic
diseases as a terminal hematologic condition and is an important
issue in managing patients. Less than 10% of myelodysplastic
syndromes (MDS) develop MF during their courses and most of
them have unfavorable prognoses.
6
The exact biology of MF in
MDS patients is still unknown, since some MDS patients show
aspects of both MDS and myeloproliferative disorders,
7
and
therefore, it is possible that MF could be associated with MDS as
one phenotype of myelproliferative disorders.
We therefore searched for the JAK2 V617F mutation in
primary and secondary MF in various hematologic diseases
using frozen marrow cells or peripheral blood mononuclear
cells from patients after obtaining their written informed
consent, using the sequence-specific primer-single molecule
fluorescence detection assay (SSP-SMFD) (Bannai M, Higuchi K,
Akesaka T, Furukawa M, Yamaoka M, Sato K, Tokunaga K.
Single-nucleotide-polymorphism gonotyping for whole-gen-
ome-amplified samples using automated fluorescence correla-
tion spectroscopy. Annal Biochem 2004; 327: 215–221). We
adopted G-T mutation when the incidence of T-substitute
reached more than 10%. Furthermore, we also confirmed the
JAK2 V617F mutation using a PCR direct-sequencing (Figure 1).
Of the MF patients associated with acute myeloid leukemia
(n ¼ 3), lymphoma (n ¼ 3), and chronic myeloid leukemia
(n ¼ 3), none showed the mutation at the time of diagnosis of
MF. Approximately 40% of ET (seven of 16 patients) and primary
MF (one of four patients) showed the JAK2 V617F mutation,
whereas six of eight patients (75%) with PV showed the
mutation. Of note is that two of six patients with MDS
terminating in MF showed the mutation at the time of MF,
while no MDS patient without MF (MDS: n ¼ 38: 20 had
refractory anemia (RA), 16 had RA excess blasts (RAEB), and two
patients had RAEB in transformation) had the JAK2 V617F
mutation (Figure 1). Our study demonstrated that MDS with MF
is sometimes associated with the JAK2 V617F mutation, while
other underlying diseases developing MF may involve other
pathways. Moreover, these findings permit speculation that
MDS patients with the JAK2 V617F mutation may be responsible
for secondary MF in MDS patients during the process of MDS
progression. Another possibility is that the JAK2 V617F mutation
DA output-1( JAK-Val 617Phe )
Fraction-G(%) ( threshold = 18.6)
Fraction-T(%) (threshold = 17.5)
SNPGT
SNPGG
60.0
50.0
40.0
30.0
20.0
10.0
0.0
0.0 10.0 20.0 30.0 40.0 50.0
160 170 180 190 200
160 170 180 190 200
b
a
Figure 1 We assessed the JAK2 V617F mutation by the sequence-
specific primer single-molecule fluorescence detection assay (SSP-
SMFD) (Bannai M, Higuchi K, Akesaka T, Furukawa M, Yamaoka M,
Sato K, Tokunaga K. Single-nucleotide-polymorphism gonotyping for
whole-genome-amplified samples using automated fluorescence
correlation spectroscopy. Annal Biochem 2004; 327: 215–221). This
technique utilizes primer extension technology combined with
fluorescent polarization. In this method, a fluorescent-labeled ddNTP
specific for the mutation is incorporated into the primer, which binds
immediately upstream from the mutation site, and the difference in
fluorescent polarization between incorporated and unincorporated dd
NTPs is detected. Percentages (horizontal axis (G) and vertical axis (T))
indicate extension efficacy of the 20 nm primer, and we adopted G-T
mutation when the incidence of T-substitute reached X10%.
Diamonds indicate patients with deviation of G-T substitute
corresponding to the JAK2 V617F mutation (a). Arrows indicate
patients with MDS with MF. PCR-direct sequencing of the comple-
mentary strand was also performed in order to confirm mutation of the
JAK2 V617F tyrosine kinase (NM_004972) in patients with myelo-
dysplastic syndrome with myelofibrosis. The PCR conditions were as
follows; preheating at 951C for 10 min, followed by 40 cycles at 951C
for 30 s, 641C for 30 s, and 721C for 1 min, and a final extension at
721C for 10 min. Reactions for direct sequencing of the PCR product
were performed with BigDye Terminator ver3.1 (Perkin-Elmer Cetus,
Fremont, CA, USA). The upper panel shows no mutation of the JAK2
V617F mutation, and the lower panel shows the C-A substitution of
the complementary reverse strand, resulting in the JAK2 V617F
mutation (G-T substitution in the forward strand). M indicates C-A
mutation of the complementary reverse strand (b).
Received 18 August 2005; accepted 9 September 2005; published
online 20 October 2005
Correspondence: Dr K Ohyashiki, First Department of Internal
Medicine, Tokyo Medical University, 6-7-1 Nishi-shinjuku, Shinju-
ku-ku, Tokyo 160-0023, Japan; Fax: þ 81 3 5381 6651;
E-mail: ohyashik@rr.iij4u.or.jp
Correspondence
2359
Leukemia

may previously exist and its clinical manifestation mimics MDS,
that is, myelodysplastic features with cytopenias, but its biologic
nature is closely associated with myeloproliferative disorders.
Unfortunately, we did not detect its mutation before MF in our
MDS/MF patients. Although MDS patients with MF have an
unfavorable prognosis, the current study demonstrates the
genotypic heterogeneity of such patients.
Acknowledgements
Thanks are due to Professor J Patrick Barron for his review of this
manuscript and Mr Kunio Hori and Tohru Makino, NovusGene,
Tokyo, for their technical assistance. This work was supported in
part by a Grant-in-Aid for ‘Intractable Hematopoietic Diseases’
from the Ministry of Health, Welfare, and Labor, Japan (to KO),
the ‘High-Tech Research Center’ Project from the Ministry of
Education, Culture, Sports, Science and Technology: MEXT) (to
KO, JHO), and by the ‘University-Industry Joint Research Project’
from MEXT (to KO, JHO).
K Ohyashiki
1
Y Aota
1
D Akahane
1
A Gotoh
1
K Miyazawa
1
Y Kimura
1
JH Ohyashiki
2
1
The First Department of Internal Medicine,
Tokyo Medical University, Tokyo, Japan; and
2
Intractable Immune System Research Center,
Tokyo Medical University, Tokyo, Japan
References
1 Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR
et al. A gain-of-function mutation of Jak2 in myeloproliferative
disorders. N Engl J Med 2005; 352: 1779–1790.
2 Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ
et al. Activating mutation in the tyrosine kinase JAK2 in
polycythemia vera, essential thrombocythemia, and agnogenic
myeloid metaplasia. Cancer Cell 2005; 7: 387–397.
3 James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C
et al. A unique clonal JAK2 mutation leading to constitutive
signalling causes polycythaemia vera. Nature 2005; 484:
1144–1148.
4 Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S
et al. Cancer genome project. Acquired mutation of the tyrosine
kinase JAK2 in human myeloproliferative disorders. Lancet 2005;
365: 1054–1061.
5 Steensma DP, Dewald GW, Lasho TL, Powell HL, McClure RF,
Levine RL et al. The JAK2 V617F activating tyrosine kinase
mutation is an infrequent event in both ‘atypical’ myeloproliferaive
disorders and myelodysplastic syndromes. Blood 2005; 106:
1207–1209.
6 Ohyashiki K, Sasao I, Ohyashiki JH, Murakami T, Iwabuchi A,
Tauchi T et al. Clinical and cytogenetic characteristics of
myelodysplastic syndromes developing myelofibrosis. Cancer
1991; 68: 178–183.
7 Ohyashiki K, Yokoyama K, Kimura Y, Ohyashiki JH, Ito Y,
Kuratsuji T et al. Myelodysplastic syndrome evolving into a
myeloproliferative disorder: one disease or two? Leukemia 1993;
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Angiogenesis and mast cells in Hodgkin lymphoma
Leukemia (2005) 19, 2360–2362. doi:10.1038/sj.leu.2403992;
published online 13 October 2005
TO THE EDITOR
Hodgkin lymphoma (HL) differs from other lymphomas because
the malignant cells, the Hodgkin and Reed–Sternberg (HRS)
cells, are in minority and the majority of the tissue consists of
surrounding benign cells, for example, eosinophilic granulo-
cytes and mast cells, fibrosis and a varying number of
microvessels. It has recently been reported that angiogenesis
correlates to poor prognosis in HL.
1
We have previously reported that HL patients with many mast
cells in their tumour tissue have a worse prognosis.
2
Mast cells
produce functionally active CD30 ligand (CD30L) and the poorer
prognosis has been proposed to be caused by a stimulation of
HRS by CD30L.
3
Furthermore, we have shown that mast cells,
upon stimulation with CD30, release cytokines and chemokines,
among which interleukin-8 (IL-8) is known to have angio-
genic properties (manuscript in preparation). In other lympho-
mas, mast cells are proposed to contribute to angiogenesis.
4
In order to increase our understanding of inflammatory cells,
their importance in tumour progression and especially angio-
genesis in HL, we investigated the possible relation between the
number of mast cells and the microvessel count in primary
diagnostic HL tissue. We also wanted to further elucidate the
prognostic implication of microvessel count in HL.
Patient samples and clinical data were acquired from the
database of the National Health Care Programme for HL in
Sweden. A total of 120 patients treated with curative intention,
according to the principles of the Health Care Programme
2
in
the Uppsala/O
¨
rebro health care region between 1989 and 1994,
were included. The paraffin-embedded tissue samples were
from HL involved lymph nodes from the primary diagnosis. The
clinical characteristics are presented in Table 1. Progression
free survival (PFS) and HL specific survival (HLS) were analysed.
The mean follow-up of living patients was 11 years (range 6–15
years).
The estimation of the number of microvessels immunohisto-
chemically stained for CD31 (Figure 1), was done by one of the
authors using the Chalkley technique.
5
Three to five fields with
the highest concentration of vessels (a hot spot) were counted
and an average of the highest three countings in every case was
used. In all, 20 cases were recounted independently by another
author and the counts correlated with an R-value of 0.75,
(P ¼ 0.0002). All evaluations were done without knowledge of
patient data. The counts varied from 1 to 12 vessels/hot spot.
The median was 3 and the 75th percentile was 4.3 vessels/hot
spot. Nonbulky disease correlated to high microvessel count
(Table 1) and there was a lower proportion of patients with
WBC415 in the upper quartile group (data not shown), but
there were no other correlations to histology, laboratory
parameters, stage, B-symptoms or sex.
In univariate analyses, HL patients with a high microvessel
count, cutoff at the 75th percentile (n ¼ 33), have a worse PFS
Received 30 August 2005; accepted 15 September 2005; published
online 13 October 2005
Correspondence: Dr I Glimelius, Department of Oncology, Radiology
and Clinical Immunology, Uppsala University Hospital, Rudbeck
laboratory C11, Uppsala S-751 85, Sweden; Fax: þ 46 18 611 34 32;
E-mail: Ingrid.Glimelius@home.se
Correspondence
2360
Leukemia