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BLOOD, 1 OCTOBER 2007 䡠 VOLUME 110, NUMBER 7
CORRESPONDENCE
lymphoblastic leukaemia with rearrangements of the 11q23 chromosomal region. Lancet. 2002;359:1909-1915.
5.
Biondi A, Cimino G, Pieters R, Pui CH. Biological and therapeutic aspects of
infant leukemia. Blood. 2000;96:24-33.
6.
Pieters R, Schrappe M, de Lorenzo P, et al. Outcome of infants less than one
2775
year of age with acute lymphoblastic leukemia treated with the Interfant-99 protocol. Lancet. 2007;370:240-250.
7.
Ozeki K, Kiyoi H, Hirose Y, et al. Biologic and clinical significance of the
FLT3 transcript level in acute myeloid leukemia. Blood. 2004;103:19011908.
To the editor:
Cytoplasmic localization of phosphorylated STAT5 in human acute myeloid leukemia
is inversely correlated with Flt3-ITD
The fms-related tyrosine kinase-3 internal tandem duplication
(Flt3-ITD) confers growth-factor independence on acute myeloid
leukemia (AML) cells and is associated with an unfavorable
prognosis. Signal transducer and activator of transcription 5
(STAT5) is a downstream mediator of Flt3 signaling in AML;
however, flow cytometry for phosphorylated STAT5 (pSTAT5) did
not show specific association with Flt3-ITD.1 Since a recent study
published in Blood by Harir et al2 reported pSTAT5 in the
cytoplasm of leukemic samples, we wanted to determine whether
this localization to the cytoplasm might be influenced by Flt3-ITD
status and whether nuclear pSTAT5 would be a more informative end point. Using a routine immunohistochemistry technique
that we previously reported for detecting activated STAT5 in
chronic myeloproliferative disease,3 we have now analyzed
pSTAT5 staining in myeloid blasts in bone marrow biopsies
from patients with AML.
STAT5 tyrosine phosphorylation was detected immunohistochemically with the anti-phosphoSTAT5a/b (Y694/99) mouse monoclonal
antibody (AX1; Advantex Bioreagents) as described by others in breast
cancer.4 In our study, we analyzed a total of 40 patients with AML for
nuclear versus cytoplasmic staining of pSTAT5 using the AX1 antibody.
Total pSTAT5 was seen in 15 of 40 patients, while the Flt3-ITD was
detected in 8 (20%) patients. Interestingly, as shown in Figure 1,
Flt3-ITD⫹ samples had predominant nuclear pSTAT5, as would be
expected from increased transcriptional activation. However, the Flt3ITD⫺ samples showed some nuclear staining, but had much more
prominent cytoplasmic pSTAT5 staining with significant variation
between groups (Kruskal-Wallis test; P ⫽ .03). A total of 2 patients
showed predominant cytoplasmic staining and no nuclear staining. The
Figure 1. Relationship between Flt3-ITD status and distribution of
phospho-STAT5 between nucleus and cytoplasm. The relative
staining intensity for pSTAT5 was determined by manually scoring slides
using a scale of 0 (neg) to 3 (⫹⫹⫹) obtained from fixed bone marrow core
biopsy of newly diagnosed patients with AML. Those scored as neg did not
have detectable pSTAT5, and those scored as 3 had the highest intensity of
staining. Shown are examples of patient samples with staining for nuclear
pSTAT5 at each level. A patient sample showing predominant cytoplasmic
pSTAT5 (⫹⫹) is shown for comparison. Flt3-ITD status was determined by
PCR on marrow clot sections using capillary electrophoresis. pSTAT5 Cy
indicates cytoplasmic; pSTAT5 Nu, nuclear; and NA, not available. Slides
were viewed with an Olympus BX50 microscope (Olympus, Center Valley,
PA) using a 50⫻/0.90 oil-immersion objective lens and pSTAT5 immunostains. Images were acquired with an Insight 11.2 color mosaic camera
(Diagnostic Instruments, Sterling Heights, MI) and processed with Spot
Basic 4.1.3 (Diagnostic Instruments) and Adobe Photoshop elements 2.0
(Adobe, San Jose, CA) software.
distribution of cytoplasmic staining was significantly different between
Flt3-ITD⫹ and Flt3-ITD⫺ patients (Mann-Whitney test; P ⫽ .04).
The study by Harir et al2 demonstrated in mouse and human
samples that STAT5 may potentially have cytoplasmic functions
that promote PI3-kinase activation of Akt. Our data confirm the
cytoplasmic localization of pSTAT5 in a larger set of patient
samples and further suggests that this localization is modulated by
the presence of Flt3-ITD. This is an important finding since it
suggests that competing biological mechanisms are responsible for
the nuclear versus cytoplasmic distribution of pSTAT5 in AML.
Cytoplasmic pSTAT5 may not be directed efficiently to the nucleus
in the absence of essential interactions provided by the Flt3-ITD
scaffold. Alternatively, the hyperphosphorylation of STAT5 mediated by Flt3-ITD may override other mechanisms that may
sequester pSTAT5 in the cytoplasm.5 This finding could be
analogous to the requirement for JAK2V617F to interact with type I
cytokine receptors to promote STAT5 transcriptional activation and
cytokine-independent growth.6 Finally, our data also demonstrate
that detection of nuclear, but not total pSTAT5 could be the most
meaningful test for rapidly identifying patients who may respond to
Flt3 or other tyrosine kinase inhibitors.
Kevin D. Bunting, Xiu Yan Xie, Ilka Warshawsky, and Eric D. Hsi
Conflict-of-interest disclosure: The authors declare no competing financial
interests.
Correspondence: Eric D. Hsi, Department of Clinical Pathology and Laboratory
Medicine, Taussig Cancer Center, The Cleveland Clinic Foundation, Case
Comprehensive Cancer Center, Cleveland, OH 44195; e-mail: [email protected].
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2776
BLOOD, 1 OCTOBER 2007 䡠 VOLUME 110, NUMBER 7
CORRESPONDENCE
JAK2 V617F mutation and provides evidence of in vivo JAK2 activation. Am J
Surg Pathol. 2007;31:233-239.
References
1.
Pallis M, Seedhouse C, Grundy M, Russell N. Flow cytometric measurement of
phosphorylated STAT5 in AML: lack of specific association with FLT3 internal
tandem duplications. Leuk Res. 2003;27:803-805.
2.
Harir N, Pecquet C, Kerenyi M, et al. Constitutive activation of Stat5 promotes
its cytoplasmic localization and association with PI 3-kinase in myeloid leukemias. Blood. 2007;109:1678-1686.
3.
Aboudola S, Murugesan G, Szpurka H, et al. Bone marrow phospho-STAT5
expression in non-CML chronic myeloproliferative disorders correlates with
4.
Nevalainen MT, Xie J, Torhorst J, et al. Signal transducer and activator of transcription-5 activation and breast cancer prognosis. J Clin Oncol. 2004;22:2053-2060.
5.
Kawashima T, Bao YC, Nomura Y, et al. Rac1 and a GTPase-activating protein,
MgcRacGAP, are required for nuclear translocation of STAT transcription factors. J Cell Biol. 2006;175:937-946.
6.
Lu X, Levine R, Tong W, et al. Expression of a homodimeric type I cytokine receptor is required for JAK2V617F-mediated transformation. Proc Natl Acad Sci
U S A. 2005;102:18962-18967.
To the editor:
Is congenital secondary erythrocytosis/polycythemia caused by activating mutations
within the HIF-2␣ iron-responsive element?
Study of inherited polycythemia/erythrocytosis has revealed defects in the oxygen-sensing pathway and provided insights into the
regulation of erythropoietin (Epo) synthesis by hypoxia inducible
factor (HIF).1 HIF is a dimer consisting of ␣ and ␤ subunits. Two
different ␣ isoforms exist: HIF-1␣, which is ubiquitously expressed, and HIF-2␣ or EPAS1, which is limited to specific tissues.
The constitutively expressed HIF-␤ subunit is not hypoxia regulated.2 In normoxia, the HIF-1␣ and HIF-2␣ proteins are rapidly
degraded through a complex interaction with one of several
iron-containing prolyl hydroxylases (PHDs), leading to the binding
of the von Hippel Lindau protein (pVHL), ubiquitination, and
degradation by the proteasome. This interaction constitutes the
Figure 1. Hypothesis for HIF-2␣ deregulation through
putative mutations present in its 5ⴕ IRE and mutational sequence screening of the 5ⴕ UTR of HIF2A.
(A) Iron and oxygen regulation of HIF-2␣ (EPAS1) in
oxygen homeostasis. Availability of iron influences the
level of HIF-induced Epo gene transcription via binding to
the 5⬘ IRE of HIF2A. In iron deficiency, the IRE is bound
by iron regulatory proteins (IRPs), which prevent HIF-2␣
translation, but when iron is replete, the IRPs cannot bind
the IRE and do not impede HIF-2␣ protein expression. In
hypoxia, HIF-2␣ is free to escape proteasomal degradation and induce Epo gene expression. The loss of
translational inhibition due to mutations in the HIF2A IRE
may result in inappropriately high HIF-2␣ levels with
deregulated Epo production, thus allowing for the development of erythrocytosis. (B) Sequence of the 5⬘ UTR
region of HIF2A. The IRE region is indicated in yellow
(nucleotides 81298 to 81330; RefSeq AC016696). Primer
sets 1 and 2 are as indicated by black and blue arrows,
respectively. All SNPs are indicated in blue. Described
SNP rs17039192 is labeled Y and located at nucleotide
81261. The 3 uncharacterized SNPs are indicated by R,
K, and R, and are located at nucleotides 81405, 81411,
and 81424. TATA box and ATG sequences are underlined.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2007 110: 2775-2776
doi:10.1182/blood-2007-05-090969
Cytoplasmic localization of phosphorylated STAT5 in human acute
myeloid leukemia is inversely correlated with Flt3-ITD
Kevin D. Bunting, Xiu Yan Xie, Ilka Warshawsky and Eric D. Hsi
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