JAK2V617F mutation and spontaneous megakaryocytic or erythroid colony formation in patients with essential thrombocythaemia (ET) or polycythaemia vera (PV)

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Leukemia Research 33 (2009) 54–59

JAK2V617F mutation and spontaneous megakaryocytic or erythroid colony formation in patients with essential thrombocythaemia (ET) or polycythaemia vera (PV) Satu Mustjoki a,∗ , Ioana Borze b , Terra L. Lasho c , Riitta Alitalo a , Animesh Pardanani c , Sakari Knuutila b , Eeva Juvonen d b

a Department of Clinical Chemistry, Laboratory of Haematology, Helsinki University Central Hospital, Helsinki, Finland Department of Pathology, Haartman Institute and HUSLAB, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland c Division of Haematology and Haematopathology, Mayo Clinic, Rochester, MN, USA d Department of Medicine, Division of Haematology, Helsinki University Central Hospital, Helsinki, Finland

Received 12 March 2008; received in revised form 7 July 2008; accepted 11 July 2008 Available online 28 August 2008

Abstract The in vitro cultures of erythroid (BFU-E) and megakaryocytic (CFU-Meg) progenitors have been useful diagnostic tools in myeloproliferative disorders (MPD). However, after the discovery of the JAK2V617F mutation, their diagnostic role has been uncertain. In this single-centre retrospective study we analyzed JAK2V617F mutation in 58 ET and 42 PV patients diagnosed according to WHO criteria and compared the results with those of colony forming assays with special emphasis on CFU-Meg growth. 91% of PV and 57% of ET patients had JAK2V617F mutation and they all showed spontaneous BFU-E growth. However, endogenous BFU-E formation was also seen in nine JAK2V617F mutation negative patients displaying also a normal JAK2 exon 12 allele. Endogeneous CFU-Meg colony formation was found in 59% of PV and 53% of the ET patients. A subgroup of ET patients (n = 7) displayed sole spontaneous CFU-Meg growth without spontaneous BFU-E growth. They all were JAK2 mutation negative, but one of them had MPL mutation. In conclusion, in vitro cultures of haematopoietic progenitors are sensitive diagnostic tools in the present group of 100 MPD patients revealing also JAK2 mutation negative ET and PV patients displaying sole spontaneous CFU-Meg or BFU-E growth. © 2008 Elsevier Ltd. All rights reserved. Keywords: ET; PV; JAK2; Colony formation; Diagnostic tool

1. Introduction The diagnosis of PV and ET has been largely based on the combination of positive diagnostic criteria, such as morphological findings in the bone marrow (BM), and exclusion of reactive or secondary erythrocytosis or thrombocytosis. The differential diagnosis may, in some cases, be problematic. The ∗ Corresponding author at: Helsinki University Central Hospital, Department of Medicine, Division of Haematology, Biomedicum Helsinki, P.O. Box 700, 00029 Helsinki, Finland. Tel.: +358 9 471 71898; fax: +358 9 471 71897. E-mail address: [email protected] (S. Mustjoki).

0145-2126/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2008.07.008

presence of spontaneous erythroid and/or megakaryocytic colony formation in in vitro cultures of haematopoietic progenitors has proved to be a good diagnostic tool confirming the presence of these disorders [1]. Recently, JAK2V617F mutation analysis has largely replaced the in vitro cultures of haematopoietic progenitors, but so far there are only a few studies where the in vitro growth pattern and the JAK2 mutation status have been compared. In addition, only the spontaneous growth of erythroid progenitors has been included in these studies. The discovery of JAK2V617F mutation has inspired an intensive search for novel mutations in V617F mutation negative patients. MPLW515L/K mutation occurring in the

S. Mustjoki et al. / Leukemia Research 33 (2009) 54–59

thrombopoietin receptor, MPL, was discovered in 2006. It leads to cytokine-independent activation of the JAK/STAT signalling pathway, but it has been shown to be present only in 5–10% of patients with ET or myelofibrosis [2,3]. Novel and even more rarely occurring mutations affecting JAK2 exon 12 were discovered last year in V617F mutation negative patients [4,5]. The aim of the present study was to compare the result of JAK2 mutation analysis and the in vitro growth pattern of haematopoietic progenitors with the special emphasis on the megakaryocytic growth pattern, which to the best of our knowledge, has not been carried out before. In addition, we further aimed to examine the role of MPL and JAK2 exon 12 mutations in patients with wild type JAK2 and spontaneous colony formation.

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analysis with G-banding method showed a clonal myeloproliferative disorders (MPD)-related cytogenetic abnormality. In addition, one patient bore a constitutional karyotype change. All six patients with a clonal disease-related abnormality were with PV. The study was conducted in accordance with the principles of the Helsinki declaration and was approved by the Helsinki University Central Hospital Ethics committee. Written informed consents were obtained from all patients. 2.2. In vitro cultures of haematopoietic progenitors As part of the routine diagnostic work, erythroid (BFU-E) and megakaryocytic (CFU-Meg) progenitors from BM aspirates were cultured as described previously [6]. Briefly, the mononuclear cells (MNC) were cultured in semisolid methyl cellulose cultures with (stimulated growth) or without (spontaneous growth) specific growth factors. The cultures were scored on day 14. Patients with at least 10 colonies/1 × 105 MNC were considered to have spontaneous CFU-Meg growth.

2. Materials and methods 2.1. Patients

2.3. JAK2 mutation analysis (V617F and exon 12 mutations)

The study population consisted of 58 patients with ET and 42 patients with PV diagnosed at Helsinki University Central Hospital during the years 2000–2006. The results of the diagnostic evaluations including the key bone marrow (BM) and blood tests and the in vitro growth pattern of haematopoietic progenitors were re-evaluated in regard to the JAK2 mutation status. Diagnoses of ET and PV were re-assessed retrospectively for the present study according to the World Health Organization classification. 13 ET patients with typical morphological characteristics in the BM aspirate samples were included in the study even though they had unavailable BM histology results. These 13 patients also fulfilled the diagnostic criteria of Polycythaemia Vera Study Group (PVSG). The female/male ratio of all patients was 49/51. At the time of the diagnosis the median age of the patients was 54 years (range 18–80 years). The laboratory data of the patients are shown in Table 1. Six out of 90 patients (7%) with an acceptable karyotype

DNA was extracted with the non-enzymatic method from frozen BM and PB samples and with the Boehringer Mannheim DNA extraction kit (Boehringer Mannheim Corporation Indianapolis, IN) from the samples in stabilization fluid. Puregene DNA purification kit (Gendra, Minneapolis, MN, USA) was used for the extraction of DNA from colony cells. The allele-specific-oligonucleotide PCR-based JAK2V617F mutation analysis was performed using stored mononuclear cells from BM (n = 80) or PB (n = 20) taken at the time of the diagnosis. The method is based on the study by Baxter et al. [7]. The sensitivity of the assay was 1%. Exon 12 mutations were analyzed with PCR sequencing using 5 -CTC CTC TTT GGA GCA ATT CA-3 forward primer and 5 GAG AAC TTG GGA GTT ATA-3 reverse primer as described previously [4].

Table 1 Association of diagnosis to laboratory, colony forming assay, and cytogenetic results in ET and PV patients Diagnosis Variable

ETa

PVa

p-Valueb

Age at dg (years) Male/Female Spontaneous CFU-Meg number of patients (%) Spontaneous BFU-E number of patients (%) JAK2 mutation positive number of patients (%) Disease-related cytogenetic abnormality (% of patients)

49 (18–80) 31/27 31/58 (53) 37/58 (64) 33/58 (57) 0/58 (0)

58 (32–80) 20/22 25/42 (59) 42/42 (100) 38/42 (91) 6/42 (14)

0.04 0.69 0.68 <0.001 <0.001 0.003

PB counts at diagnosis Haemoglobin (g/L) Haematocrit (%) Platelet count (109 /L) White blood cell count (109 /L) Erythrocyte count (1012 /L)

143 (93–183) 42 (28–54) 716 (421–1424) 7.5 (2.5–22.2) 4.8 (2.7–6.13)

176 (132–231) 52 (42–69) 497 (170–1192) 10.1 (4.2–21.6) 5.8 (4.1–8.4)

<0.001 <0.001 <0.001 0.003 <0.001

Abbreviations: ET, essential thrombocythaemia; PV, polycythaemia vera; dg, diagnosis; PB, peripheral blood; BFU-E, burst forming unit-erythroid; CFU-GM, colony forming unit-granulocyte macrophage; CFU-Meg, colony forming unit-megakaryocyte. a Continuous variables are expressed as median (range). b Kruskal–Wallis test for continuous variables and Fisher’s Exact test for categorical variables.

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2.4. MPL mutation analysis Genotyping of the MPL515 allele was performed using a LightCycler (Roche Applied Bioscience, Indianapolis, IN) assay described previously [2]. Samples with a positive signal were sequenced to confirm the finding. The sensitivity of the assay was found to be 3–5%. 2.5. Statistical analysis The evaluation of the data was done using the SPSS software program (version 14.0 for Windows, SPSS Inc). Fisher’s exact test, Kruskall–Wallis non-parametric test and Spearman rank correlation (rho) tests were used for comparison of the results. The results were considered statistically significant at p ≤ 0.05.

3. Results 3.1. Growth pattern of haematopoietic progenitors and JAK2 mutation status Spontaneous CFU-Meg growth was seen in 55% of all the patients, in 53% of the patients with ET, and in 59% of those with PV (p = 0.68) (Table 1). Spontaneous BFU-E colony formation was seen in 78% of all the patients, in 64% of the ET patients and in all PV patients (p < 0.001) (Table 1). Spontaneous CFU-Meg or BFU-E growth or both was seen in 85/100 patients with MPD. Of the present patients with MPD 71/100 (71%) were found to have JAK2V617F mutation, 57% of the ET patients and 91% of the PV patients (p < 0.001) (Table 1). In ET, the median values of the erythrocyte count, haemoglobin (Hb) concentration, and haematocrite were significantly higher in the patients with JAK2 mutation than in those with the wild type JAK2 (Table 2). The presence of JAK2 mutation was also associated with gender in the ET patients (Table 2). Of the patients with JAK2 mutation 67% were male while of those with the wild type JAK2 only 36% were male (p = 0.03).

Of the PV patients only 9% showed the wild type JAK2. As in ET, the JAK2 mutation positive PV patients had a higher erythrocyte count (5.8 × 1012 /L vs. 5.4 × 1012 /L), Hb (176 g/L vs. 159 g/L), and haematocrite values (53% vs. 48%) than those with the wild type allele, though no statistical significance was reached.

3.2. Association of JAK2 mutation with the spontaneous CFU-Meg and BFU-E colony formation Of all 71 patients with JAK2 mutation 45 (63%) had spontaneous CFU-Meg colony formation. In ET, the spontaneous CFU-Meg colony formation was seen significantly more often in the JAK2 mutated patients than in those with the wild type JAK2 (70% vs. 32%, p = 0.007, Table 2). In PV, spontaneous CFU-Meg growth was seen as often in the patients with JAK2 mutation (60% vs. 50%) as in those with the wild type allele, but the number of patients with normal JAK2 was too low for any definitive conclusions. The presence of spontaneous erythroid growth and JAK2 mutation correlated nearly completely. Spontaneous BFU-E growth was seen in all JAK2 mutation positive patients with one exception (Table 2). This patient was diagnosed as having ET, but she developed anaemia and had an excess of ring sideroblasts in the bone marrow. She showed neither spontaneous nor EPO stimulated BFU-E colony formation. The apparent diagnosis of the patient was JAK2 mutation positive refractory anaemia with ringed sideroblasts and marked thrombocytosis, RARS-T, as described recently [8]. Of the patients with the wild type JAK2 allele 9/29 (31%) showed spontaneous BFU-E growth (five ET and four PV patients) (Table 3). Samples from six of these patients (four ET and two PV) were evaluable in the exon12 mutation analysis and they all had the wild type allele (Table 3). In the EPO-stimulated cultures the numbers and morphology of BFU-E were similar in the mutated and unmutated patients.

Table 2 Association of JAK2 mutation status to laboratory and colony forming assay results in ET patients JAK2 mutation status Variable

Mutateda

Unmutateda

p-Valueb

Age at dg (years) Male/Female Spontaneous CFU-Meg number of patients (%) Spontaneous BFU-E number of patients (%)

49 (25–80) 22/11 23/33 (70%) 32/33 (97%)

55 (18–74) 9/16 8/25 (32%) 5/25 (20%)

0.87 0.03 0.007 <0.001

PB counts at diagnosis Haemoglobin (g/L) Haematocrit (%) Platelet count (109 /L) White blood cell count (109 /L) Erythrocyte count (1012 /L)

149 (93–183) 44 (28–54) 692 (421–1391) 8.6 (4.2–21.2) 5.0 (2.7–6.1)

136 (100–165) 41 (31–48) 756 (457–1424) 6.9 (2.5–22.2) 4.5 (3.1–5.5)

0.012 0.006 0.15 0.12 0.005

Abbreviations: ET, essential thrombocythaemia; dg, diagnosis; PB, peripheral blood; BFU-E, burst forming unit-erythroid; CFU-GM, colony forming unitgranulocyte macrophage; CFU-Meg, colony forming unit-megakaryocyte. a Continuous variables are expressed as median (range). b Kruskal–Wallis test for continuous variables and Fisher’s Exact test for categorical variables.

Table 3 Characteristics of JAK2 negative ET and PV patients with spontaneous BFU-E growth Dg

Gender

Age

WBC (109 /L)

Hct (%)

Hb (g/L)

Platelets (109 /L)

V617F/Exon12

BFU-Ea stim

BFU-Ea spont

CFU-GMa

CFU-Mega spont

Pat. 1 Pat. 2 Pat. 3 Pat. 4 Pat. 5 Pat. 6 Pat. 7 Pat. 8 Pat. 9

PV PV PV PV ET ET ET ET ET

M M F F F F F F M

71 64 32 52 60 24 56 55 48

8.9 43.0 11.8 7.9 13.5 8.4 7.2 15.4 6.9

55 63 49 49 41 42 42 40 48

187 198 161 165 140 139 136 123 165

198 923 321 489 638 677 1074 716 571

Negative/Negative Negative/NE Negative/Negative Negative/NE Negative/NE Negative/Negative Negative/Negative Negative/Negative Negative/Negative

114 60 138 121 105 161 79 108 187

10 28 32 15 11 16 5 22 19

20 40 71 46 39 122 29 3 77

14 15 7 6 3 13 0 5 10

Abbreviations: PV, polycythaemia vera; ET, essential thrombocythaemia; dg, diagnosis; M, male; F, female; WBC, white blood cell, Hct, haematocrit; Hb, haemoglobin; NE, not evaluable; BFU-E, burst forming unit-erythroid; CFU-GM, colony forming unit- granulocyte macrophage; CFU-Meg, colony forming unit-megakaryocyte; stim, stimulated growth; spont, spontaneous growth. a Number of colonies per culture plate.

Table 4 Characteristics of ET patients with CFU-Meg growth only Patient code

Age

WBC (109 /L)

Hb (g/L)

Platelets (109 /L)

JAK2 mutation

MPL mutation

BFU-Ea stim

BFU-Ea spont

CFU-GMa

CFU-Mega spont

Pat. 1 Pat. 2 Pat. 3 Pat. 4 Pat. 5 Pat. 6 Pat. 7

18 34 33 61 55 73 55

6.9 6.5 5.7 6.8 11.1 21.9 4.9

124 153 135 135 136 123 132

823 756 703 1424 1323 918 682

Negative Negative Negative Negative Negative Negative Negative

Negative Negative Negative Negative Negative Positive Negative

73 214 148 110 128 157 150

0 0 0 1 0 0 0

14 39 68 28 36 37 19

13 24 44 26 16 79 48

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Patient code

Abbreviations: ET, essential thrombocythaemia; WBC, white blood cell; Hb, haemoglobin; BFU-E, burst forming unit-erythroid; CFU-GM, colony forming unit-granulocyte macrophage; CFU-Meg, colony forming unit-megakaryocyte; stim, stimulated growth; spont, spontaneous growth. a Number of colonies per culture plate.

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3.3. MPLW515L mutation in patients with wild type JAK2 allele and spontaneous CFU-Meg colony formation MPL mutation was studied from the MNCs of seven JAK2V617F mutation negative ET patients who showed spontaneous CFU-Meg growth but no spontaneous BFU-E colonies. One patient was found to have MPL W515L mutation (Table 4). From one ET patient (patient 7, Table 4), in addition to blood MNCs, the MPL mutation was also analyzed from the spontaneous CFU-Meg colonies, EPO stimulated BFU-E colonies, and CFU-GM colonies. Only the wild type MPL gene was found in all these samples. Furthermore, four patients with undefined MPD and two patients with myelofibrosis who were JAK2 negative and had clear spontaneous CFU-Meg growth were tested for MPL mutation. They all were found to have the wild type allele.

4. Discussion The present and our previous studies have shown that a majority (85%) of the patients with ET or PV has either spontaneous BFU-E or CFU-Meg colony formation or both in in vitro cultures of haematopoietic progenitors [1]. JAK2V617F mutation was found in about 70% of the present patients, i.e. in most patients with PV and in about half of those with ET, which is in accordance with previous studies [7,9–11]. In ET, spontaneous CFU-Meg growth correlated statistically significantly with the presence of JAK2V617F mutation. In addition, there were 11/25 ET patients with the wild type JAK2 together with an abnormal in vitro colony formation. With one exception all patients with JAK2V617F mutation showed spontaneous BFU-E growth, but spontaneous erythroid growth was also seen in 9/29 of the patients with the wild type JAK2 allele. Altogether, in 15 patients with the wild type JAK2 allele, the spontaneous in vitro colony formation confirmed the diagnosis of MPD. In recent years the JAK2 mutation analysis has proved an important diagnostic test in MPDs. The test has many advantages; it is a PCR-based assay available in most cytogenetic laboratories, and the analysis can be performed from peripheral blood. Before the era of JAK2 mutation, for about 2 decades, spontaneous erythroid or megakaryocytic colony growth in in vitro cultures of haematopoietic progenitors has been a very useful diagnostic test in MPDs. The discovery of JAK2 V617F mutation has not reduced the diagnostic reliability of the in vitro cultures. The culture assays, however, carry several problems such as poor availability and need for a considerable know-how of cell culturing techniques. Nevertheless, also in the revised WHO diagnostic algorithm for PV, the in vitro cultures of haematopoietic progenitors have maintained their position as a minor criteria [12]. Besides JAK2V617F mutation, there are evidently also other pathogenetic events causing MPD and

resulting in abnormal in vitro colony formation. In several studies, including the present one, patients with the normal JAK2 allele have shown abnormal colony formation [10,13]. Studies comparing the in vitro growth pattern of haematopoietic progenitors and JAK2 mutation status in MPD are, so far, scarce and do not contain the data of CFUMeg [10,13,14]. Moreover, patients with the normal JAK2 allele presenting with spontaneous colony formation are the most interesting group of MPD patients in view of finding novel mutations. According to the experience of the present authors, spontaneous CFU-Meg colony formation is a specific phenomenon in MPD, but the mechanisms behind it are, at least in some cases, unclear. JAK2V617F mutation has recently been shown to be present in stem cells and megakaryocytes, but V617F mutation may not be the only explanation for abnormal megakaryocytic growth. None of the present patients with spontaneous CFU-Meg colony formation and without spontaneous BFU-E growth were JAK2 mutation positive. Also in a study by Boissinot et al. [15], JAK2 negative patients displaying endogenous CFU-Meg growth has been described. A mutation in the thrombopoietin receptor, MPLW515L mutation, has been recently discovered in some patients with ET and MF [2,3]. In the murine model, MPL mutation induced megakaryocyte hyperplasia leading to thrombocytosis [3]. We, therefore, speculated that MPL mutation may be a causative event in spontaneous CFU-Meg colony formation. However, in the present material of 13 MPD patients showing clear spontaneous CFU-Meg growth, only one patient had MPLW515L mutation. This warrants the need for further studies to define genetic alterations behind CFU-Meg growth. In conclusion, the in vitro cultures of haematopoietic progenitors are still reliable and sensitive methods in the diagnosis of ET and PV. They give additional diagnostic information especially in MPD patients showing wild type JAK2. By using these methods it is possible to find JAK2 negative patients displaying only spontaneous CFU-Meg growth and the diagnosis of MPD (especially ET) can be based on a biological assay, not only on clinical findings and exclusion criteria.

Conflicts of interest The authors report no conflicts of interest.

Acknowledgements This work was supported by the Finnish special governmental subsidy for health sciences, research and training and the Blood Disease Foundation. The expert technical assistance of the personnel in the Stem Cell Laboratory, HUSLAB, Helsinki University Central Hospital is highly appreciated.

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Authorship: SM and EJ wrote the paper. SM, IB, TL, RA, AP, SK, and EJ participated in conception and design of the study, analysis of the data and performed research.

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