CASE REPORT

Activated STAT1 and STAT5 transcription factorsin extramedullary hematopoietic tissue in a polycythemiavera patient carrying the JAK2 V617F mutation

Thomas Meyer • Volker Ruppert • Christian Gorg •

Andreas Neubauer

Received: 28 May 2009 / Revised: 28 October 2009 / Accepted: 19 November 2009 / Published online: 16 December 2009

� The Japanese Society of Hematology 2009

Abstract The somatic V617F mutation in the Janus kinase

(JAK) 2 gene, which causes a valine to phenylalanine substi-

tution at position 617, has recently been found in the majority

of patients with polycythemia vera and in many cases with

essential thrombocythemia or idiopathic myelofibrosis. Here,

we report on a 76-year-old female patient presenting with

JAK2V617F-positive polycythemia vera and a pelvic mass

with extramedullary hematopoiesis. Immunohistochemistry

demonstrated tyrosine phosphorylation of JAK2 kinase as well

as STAT1 and STAT5 transcription factors. However, only a

minority of the total STAT1 pool was tyrosine-phosphorylated

and, in contrast to its unphosphorylated counterpart,

phospho-STAT1 clearly showed nuclear accumulation. While

megakaryotes expressed virtually no phospho-STAT1, phos-

phorylated STAT5 was mainly restricted to megakaryocytes

and rarely detected in non-megakaryocytes. Our data suggest

that dysregulated STAT signal pathways are engaged in

extramedullary hematopoiesis in polycythemia vera.

Keywords Polycythemia vera � STAT proteins �Extramedullary hematopoiesis

1 Introduction

In more than 95% of polycythemia vera patients and

approximately half of the patients with essential

thrombocythemia and idiopathic myelofibrosis a clonal

mutation in the pseudokinase domain of the tyrosine

kinase JAK2 was identified that renders the unmutated

kinase domain constitutively active [1, 2]. Exchange of a

critical phenylalanine for valine at position 617 in the

JH2 domain of JAK2 results in dysregulated JAK2 sig-

naling that occurs in hematopoietic stem cells and pre-

disposes toward erythroid differentiation [3, 4]. The

clinical relevance of the JAK2V617F mutation in the

pathogenesis of polycythemia vera was tested in a mouse

bone marrow transplant model [1]. Transplantation of

mouse marrow cells transduced with the JAK2V617F

mutant allele into lethally irradiated recipients resulted in

erythrocytosis typical of polycythemia vera [1]. More-

over, mice transplanted with JAK2V617F-expressing

cells developed a polycythemia vera-like disease that

progressed to myelofibrosis in a manner analogous to the

human disease [5]. Potential downstream targets for

JAK2 include members of the signal transducer and

activation of transcription (STAT) family of transcription

factors, which upon JAK2 activation become phosphor-

ylated on a signature tyrosine residue in their carboxy

termini. After translocation into the nucleus, tyrosine-

phosphorylated STAT dimers bind to palindromic

sequences in the promoters of cytokine-stimulated genes

and modulate their expression. In bone marrow biopsies

from patients with polycythemia vera, Teofili and col-

leagues [6] have recently demonstrated increased phos-

pho-STAT3 and phospho-STAT5 expression, which

differed from patients with essential thrombocythemia

and idiopathic myelofibrosis. In this article we report

on our immunohistochemical findings of JAK2, STAT1

and STAT5 activation in a patient presenting with

polycythemia vera and massive extramedullary

hematopoiesis.

T. Meyer (&) � V. Ruppert

Department of Cardiology, University of Marburg,

Baldingerstraße 1, 35033 Marburg, Germany

e-mail: meyert@med.uni-marburg.de

C. Gorg � A. Neubauer

Department of Hematology and Oncology,

University of Marburg, Marburg, Germany

123

Int J Hematol (2010) 91:117–120

DOI 10.1007/s12185-009-0457-4

2 Case report

2.1 Clinical history

A 76-year-old female patient presented with shortness of

breath on exertion, fatigue, and an abdominal distension

with fullness and mild pain. The patient had a history of

polycythemia vera for the last 20 years and was treated with

hydroxycarbamide and phlebotomies. Splenomegaly was

diagnosed and the spleen irradiated with 10 Gray. Three

years after radiotherapy, splenectomy was performed fol-

lowing a traumatic spleen rupture. In 2006, the JAK2V617F

mutation was identified using PCR amplification and DNA

sequencing. Physical examination at admission revealed a

palpable mass in the pelvis. Abdominal ultrasonography

demonstrated a well-circumscribed, homogeneous, echo-

genic, and spherically shaped mass in the pelvis adjacent to

the iliac vessels (Fig. 1). A computed tomography scan of

the abdomen confirmed a homogeneous mass that was

consistent with a conglomerate of hypertrophic lymph nodes

or extramedullary hematopoiesis. At presentation, she had a

hematocrit of 34%, a remarkable leukocytosis (21 9 109/l),

a platelet count of 193 9 109/l and an elevated serum lactate

dehydrogenase activity (620 U/l). A bone marrow aspirate

showed no signs of transformation into acute myeloid leu-

kemia. The patient underwent an ultrasound-guided biopsy

of the pelvic mass. The biopsy specimens were fixed in

formalin and embedded in paraffin for further analyses.

2.2 Immunohistochemical results

Sections were cut and processed for immunohistochemical

analyses after their deparaffinization and rehydration.

Conventional stainings of the biopsy specimen showed

evidence of extramedullary hematopoiesis as judged by the

presence of precursor cells from erythroid, myeloid, and

megakaryocytic lineages (Fig. 2a). Various components of

granulopoiesis including immature precursor cells were

identified by their immunoreactivity for chloroacetate

esterase and myeloperoxidase (Fig. 2b, c). Numerous

megakaryocytes and small lymphocytes with a thin rim of

cytoplasm were detectable confirming extramedullary

hematopoietic activity. CD34-positive cells were scattered

throughout the biopsy sample (Fig. 2d). Tyrosine-phos-

phorylated JAK2 was detected in small cells (Fig. 2e).

Both STAT1 and STAT5 were abundantly expressed in the

biopsy sample with the exception of megakaryocytes,

which virtually lacked STAT1 expression (Fig. 2f, g). In

the pelvic mass, tyrosine-phosphorylated STAT1 was

detected in isolated small cells, where it was typically

restricted to the nucleus indicative of its nuclear

accumulation (Fig. 2h). In a high-power field, 29 ± 5

phospho-STAT1-positive cells were counted. In contrast,

in immunohistochemically stained tissue samples obtained

from the explanted spleen tyrosine-phosphorylated STAT1

was below the detection threshold (Fig. 2i). Staining with a

polyclonal antibody specifically recognizing tyrosine-

phosphorylated STAT5 demonstrated clear immunoposi-

tivity in the extramedullary hematopoietic tissue of the

pelvic mass (Fig. 2j). We counted a mean number of

54 ± 3 phospho-STAT5-positive cells per high-power

field. In contrast to phospho-STAT1, activated STAT5 was

highly expressed in megakaryocytes but was also detected,

albeit to a lesser extent, in non-megakaryocytes.

3 Discussion

Immunohistochemical analyses of bone marrow biopsies

have been routinely used in the differential diagnosis of

patients presenting with myeloproliferative diseases.

Biopsies from polycythemia vera patients display a trilin-

eage hyperplasia, affecting the erythroid, myeloid, and

megakaryocytic lineages, which results in an increased

peripheral red blood cell mass, frequently accompanied by

elevated peripheral granulocyte and platelet counts. Since

the identification of a single amino acid exchange from

valine to phenylalanine in position 617 in the majority of

polycythemia vera patients, genotyping of the mutation in

exon 12 of the JAK2 gene has become an useful tool in the

diagnosis of myeloproliferative diseases [7]. However,

despite the central role of the JAK2V617F mutation in the

pathogenesis of polycythemia vera, much less is known on

the contribution of downstream targets of the dysregulated

JAK signal pathway that are required for cellular trans-

formation [6, 8, 9]. One report has described increased

Fig. 1 Ultrasonographical image of a pelvic mass in a 76-year-old

female patient presenting with JAK2V617F-positive polycythemia

vera

118 T. Meyer et al.

123

phosphorylation levels of both STAT3 and STAT5 in bone

marrow biopsies obtained from 54 patients with polycy-

themia vera [6]. This pattern of STAT activation was dif-

ferent from the increased phospho-STAT3 and reduced

phospho-STAT5 expression in the bone marrows from

patients with essential thrombocythemia and the reduced

phosphorylation of both STAT proteins in patients with

idiopathic myelofibrosis [6].

In contrast to bone marrow, expression of STAT pro-

teins in extramedullary hematopoietic tissue has not been

investigated so far. In polycythemia vera patients extra-

medullary hematopoiesis is a typical clinical symptom that

frequently results in the formation of single or multiple

tumor masses. In the case report presented, we tested the

expression pattern of two STAT proteins, namely STAT1

and STAT5, in extramedullary hematopoietic tissue by

means of immunohistochemistry. Here, we show that both

STAT1 and STAT5 were abundantly expressed in the

extramedullary hematopoietic mass. Tyrosine-phosphory-

lated STAT1 and STAT5, however, were detected only in a

minority of cells and differed in their cellular distribution.

Megakaryocytes were generally lacking phospho-STAT1

expression, but typically displayed a positive staining for

tyrosine-phosphorylated STAT5. Phospho-STAT1 was

detected in the nuclei of small non-megakaryocytic cells

scattered throughout the biopsy section. Nuclear accumu-

lation of STAT1 is indicative of its activation, thus con-

firming the presence of transcriptionally active STAT1

dimers in the extramedullary hematopoietic mass. In

contrast, tyrosine-phosphorylated STAT5 was expressed

predominantly in megakaryocytes and much less in non-

megakaryocytes. Thus, the cellular distribution in the

parailiac mass differed between phosphorylated STAT1

and STAT5, but was similar to that described in the bone

marrow of polycythemia vera patients [6, 8].

Somatic mutations in the JAK2 gene, particularly the

V617F mutation, result in dysregulated JAK-STAT signal

pathways, which had been implicated in the pathophysi-

ology of polycythemia vera. For the rare patients who do

not harbor the V617F substitution, exon 12 JAK2 mutations

were also discovered to result in activated forms of the

kinase [7]. However, it is unclear how constitutively acti-

vated JAK2 leads via dysregulated STAT signaling to a

clonal disorder of hematopoietic progenitor cells. STAT1

can induce apoptotic cell death and interferon stimulation

exerts anti-proliferative effects [10]. Therefore, it is con-

ceivable that STAT1 counteracts the overwhelming pro-

liferative response which results from malignant

transformation of hematopoietic stem cells. STAT1 may

afford protection by activating the expression of pro-

apoptotic genes, rather than playing a direct and causative

role in the pathogenesis of the disease. In contrast to the

function of STAT1 as a crucial regulator of cell death,

constitutive activation of STAT5 via phosphorylated JAK2

contributes directly to ligand-independent cell growth and

promotes cell proliferation [11]. Thus, activation of STAT1

and STAT5 proteins in the extramedullary tumor mass may

mediate opposing effects on cell proliferation and disease

progression.

Taken together, we detected activated STAT1 and

STAT5 proteins in a JAK2V617F-positive polycythemia

vera patient presenting with extramedullary hematopoiesis

in a parailiac mass. Our immunohistochemical data showed

that only a small amount of the total STAT pool is tyrosine-

phosphorylated and that the cellular distribution of STAT1

and STAT5 differed substantially.

Fig. 2 Immunohistochemical stainings of a pelvic mass (a–h, j) and

the spleen (i) in a polycythemia vera patient showing evidence of

extramedullary hematopoiesis and STAT expression. Sections were

stained with hematoxylin–eosin (a), chloroacetate esterase (b),

myeloperoxidase (c), CD34 (d), phosphorylated JAK2 (e), STAT1

(f), STAT5 (g), tyrosine-phosphorylated STAT1 (h, i), and tyrosine-

phosphorylated STAT5 (j), respectively. Note the presence of

phospho-JAK2 and phospho-STAT1 in extramedullary hematopoiesis

(h) and its absence in splenic tissue (i). Phospho-STAT5 expression

was predominantly detected in extramedullary megakaryotes (j)

STAT proteins in polycythemia vera 119

123

Acknowledgments We thank Marlies Crombach for excellent

technical assistance. The study was supported by a grant from the

Deutsche Krebshilfe to T.M.

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