Megakaryocytic Morphology and Clinical Parameters in Essential Thrombocythemia, Polycythemia Vera, and Primary Myelofibrosis With and Without JAK2 V617F Nataliya Vytrva, MD; Elvira Stacher, MD; Peter Regitnig, MD; Wilma Zinke-Cerwenka, MD; Sabine Hojas, MD; Eva Hubmann, MD; Anna Porwit, MD, PhD; Magnus Bjorkholm, MD, PhD; Gerald Hoefler, MD; Christine Beham-Schmid, MD

 Context.—Megakaryocytes are the ‘‘hallmark’’ of Philadelphia chromosome–negative myeloproliferative neoplasms, such as essential thrombocythemia, polycythemia vera, and primary myelofibrosis; their morphology in correlation with Janus kinase 2 (JAK2 V617F) mutation as well as clinical and laboratory parameters remains unknown. Objective.—To assess the morphology of megakaryocytes in bone marrow biopsies of patients with and without JAK2 V617F mutation. Design.—Assessment of morphologic features of megakaryocytes in 112 bone marrow biopsies (52 essential thrombocythemia, 38 polycythemia vera, and 22 primary myelofibrosis) and correlation with clinical and laboratory data. Results.—JAK2 V617F mutation was detected in 24 of 52 essential thrombocythemia cases (46.2%), 36 of 38 polycythemia vera cases (97.5%), and 14 of 22 primary myelofibrosis cases (63.6%). By investigating morphometric and clinical parameters using multivariate analysis, we

found that higher hemoglobin concentration, higher white blood cell counts, and lower platelet counts were significantly associated with JAK2 V617F. Striking morphologic similarities were found between polycythemia vera JAK2 V617F and primary myelofibrosis JAK2 V617F concerning the localization and cytoplasmic size of megakaryocytes. Although polycythemia vera JAK2 V617F and essential thrombocythemia JAK2 V617F shared similarities in localization, distribution, and amount of megakaryocytes, morphology was different. Megakaryocytic morphology also differed between primary myelofibrosis JAK2 V617F and essential thrombocythemia JAK2 V617F. Conclusion.—Our results indicate that primary myelofibrosis JAK2 V617F and polycythemia vera JAK2 V617F share pathogenetic pathways, resulting in morphologically similar megakaryocytes. (Arch Pathol Lab Med. 2014;138:1203–1209; doi: 10.5858/arpa.2013-0018-OA)

T

(PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF).1,2 In 2005 James et al3 described a specific acquired point mutation in the Janus kinase 2 gene (JAK2) in PV. Soon after, several other groups detected this mutation in PV as well as in other Ph MPNs.4–7 JAK2 mediates the transmission of signals from cytokine receptors to multiple signal transduction pathways that regulate cell survival, proliferation, differentiation, and apoptosis.8,9 The most prevalent JAK2 gene mutation is characterized by a point mutation (G-to-T transversion) resulting in a valine-to-phenylalanine substitution in the JH2 domain at the amino acid position 617 (V617F) of the Jak2 protein. As a result of this mutation, Jak2 protein activates multiple downstream signaling pathways affecting gene transcription, leading to a proliferation of hematopoietic cells.3–7,10 As summarized by Hussein et al,11 JAK2 V617F occurs in 82% of PV cases (range, 65%– 97%), 53% of PMF cases (range, 35%–95%) and 49% of ET cases (range, 23%–75%). In the small number of PV cases lacking the JAK2 V617F mutation, another activating JAK2 exon 12 mutation can be identified in most cases.2,12 Because JAK2 V617F is present in only a subset of Ph MPNs, we studied whether JAK2 V617F is associated with specific morphologic bone marrow (BM) features, with a

he term Philadelphia chromosome-negative myeloproliferative neoplasms (Ph MPNs) is commonly used as a broad term for MPNs that are BCR-ABL negative; the most frequent entities under this rubric are polycythemia vera

Accepted for publication November 27, 2013. From the Institute of Pathology (Drs Vytrva, Stacher, Regitnig, Hoefler, and Beham-Schmid) and the Division of Hematology, Department of Medicine (Dr Zinke-Cerwenka), Medical University of Graz, Graz, Austria; the Division of Hematology, Department of Medicine, General Hospital Furstenfeld, Furstenfeld, Austria (Dr ¨ ¨ Hojas); the Division of Hematology, Department of Medicine, General Hospital Leoben, Leoben, Austria (Dr Hubmann); and the Department of Pathology, Radiumhemmet (Dr Porwit), and the Division of Hematology, Department of Medicine (Dr Bjorkholm), Karolinska University Hospital, Stockholm, Sweden. Dr Vytrva is now with the Department of Clinical Pathology, Ryhov Hospital, Jonk Sweden. Dr Porwit is now with the Department of ¨ oping, ¨ Laboratory Hematology, General Hospital of Toronto, Toronto, Ontario, Canada. Drs Vytrva and Stacher contributed equally to the study. The authors have no relevant financial interest in the products or companies described in this article. Reprints: Christine Beham-Schmid, MD, Institute of Pathology, Medical University of Graz, Auenbruggerplatz 25, A-8036 Graz, Austria (e-mail: [email protected]). Arch Pathol Lab Med—Vol 138, September 2014

Megakaryocytic Morphology and JAK2 Mutation Status—Vytrva et al 1203

particular focus on megakaryocytes, morphologically the ‘‘hallmark’’ component in MPNs. In addition, we studied whether there were similar morphologic features in BM and clinical characteristics within JAK2 V617F or JAK2 wild-type (JAK2 WT) cases that could reveal potential pathogenic relationships among Ph MPN subgroups. JAK2 mutational status has gained importance recently because drugs targeting JAK2 activation are being studied in clinical trials, with one being approved by the Food and Drug Administration.13 MATERIALS AND METHODS A total of 112 BM biopsies from patients with an initial diagnosis of Ph MPNs were analyzed for JAK2 mutation status. All biopsies were reevaluated and classified according to the World Health Organization 2008 criteria2 in a blinded fashion without knowledge of the JAK2 mutational status. Ten BM biopsies were included as a control group. These biopsies were taken for lymphoma staging and lacked neoplastic infiltration as well as any other obvious pathologic features. The study was approved by the local ethics committee (Votum No. 23–234). Two trephine biopsies were taken from the posterior iliac crest after obtaining written informed consent. One biopsy specimen was fixed in Carnoy solution and embedded in methyl-methacrylate.14 Consecutive 3- to 4-lm-thick sections were stained with hematoxylin-eosin, periodic acid–Schiff reagent, Prussian blue iron stain, Gomori reticulin stain, toluidine blue and May-Grunwald¨ Giemsa, respectively. The second biopsy was sent unfixed in RPMI 1640 medium (GIBCO/Invitrogen, Carlsbad, California) and stored in liquid nitrogen for further molecular analyses.

JAK2 Mutation Analysis For detection of JAK2 V617F, frozen BM specimens were used. Homogenization was performed using MagNA Lyser Green Beads (Roche Diagnostics, Mannheim, Germany) with 1 mL of lysis buffer containing Proteinase K (250 mg; Roche Diagnostics), 2.25 mL of TNE buffer, and 125 lL of 20% sodium dodecyl sulfate. Sample disruption was performed 3 times, each time at 614g for 30 seconds. Proteinase K digestion at 428C was performed for 16 hours, followed by phenol extraction and precipitation with sodium acetate and absolute ethanol. The air-dried pellet was resuspended in TNE buffer. DNA concentration was determined photometrically. DNA from BM biopsies was amplified using conditions optimized for denaturing high-performance liquid chromatography. Genomic DNA (50, 100, and 150 ng) was amplified in a 50-lL reaction volume containing 2.5 units of Optimase ProofreadingTaq (Transgenomic, Glasgow, United Kingdom), 5 lL of 103 reaction buffer containing 15 mmol of MgSO4 (Transgenomic), 0.5 lL of 200-lmol/L dinucleotide triphosphates (Pharmingen, San Diego, California), and 0.25-lmol/L each primer (MWG, Ebersberg, Germany). Primer sequences were selected using software available at http://www.mutationdiscovery.com: JAK2 V617F forward, 5 0 -AGC AGC AAG TAT GAT GAG CAA GC-3 0 ; and JAK2 V617F reverse, 5 0 -AAT ACT CTA TTG TTT GGG CAT TGT-3 0 were used to amplify a 306-bp-long segment of exon 14 of the JAK2 gene. The reaction mixture was incubated in a thermocycler (2720, Applied Biosystems, Singapore) at 958C for 5 minutes, followed by 35 cycles of 958C for 45 seconds, 588C for 45 seconds, and 728C for 45 seconds, and a final extension step at 728C for 7 minutes. To form heteroduplexes, polymerase chain reaction products were heated to 958C, and a touchdown reaction of 958C to 658C (18C per cycle) for 30 seconds was performed. In each run a WT and a mutation-positive control sample were included. Finally, the amplicon was analyzed by denaturing high-performance liquid chromatography on a Transgenomic WAVE System 3500 equipped with a DNASep cartridge. Navigator software (version 2.0.0, Transgenomic, Omaha, Nebraska) was used to determine the optimal run conditions, resulting in a melting temperature of 57.18C, a flow rate of 0.9 mL/min, and a time shift of þ1 minute. 1204 Arch Pathol Lab Med—Vol 138, September 2014

Samples were considered mutated when a distinct prepeak was detected. The detection limit of this technique was determined as 5% of mutated cells.

Semiquantitative Analysis Methyl-methacrylate–embedded biopsies were investigated semiquantitatively using a standard bright-field microscope. Ageadjusted total cellularity and hematopoietic cell lineages were categorized as decreased, normal, or increased.15 The amount of iron deposits was categorized as absent, decreased (trace), present, or increased (abundant).16 Myelofibrosis was graded as per the World Health Organization classification.2 The percentage of megakaryocytes grouped in clusters (5 or more megakaryocytes having direct contact with cell membranes) and located in contact with trabeculae, intertrabecularly; ‘‘naked’’ megakaryocytic nuclei without appreciable cytoplasm; and megakaryocytes with emperipolesis were recorded.

Morphometric Analysis A total of 7563 megakaryocytes were analyzed. Images at high power (4003) were captured to analyze 60 megakaryocytes per May-Grunwald-Giemsa section. In 9 cases this number could not ¨ be achieved (range, 16–49 megakaryocytes) because of the size of the biopsies. The open-source software ImageJ (version 1.41a, National Institutes of Health, Bethesda, Maryland) was used for morphometry.17 Megakaryocytes were outlined manually to delineate cytoplasmic and nuclear borders. Parameters analyzed included cytoplasmic as well as nuclear area (lm2), cytoplasmic and nuclear perimeters (lm), nuclear to cytoplasmic area ratio, cytoplasmic circularity (this parameter ranges between 0 and 1, with 1 representing a perfectly round circle), nuclear circularity, cytoplasmic and nuclear major diameters (lm), and cytoplasmic and nuclear minor diameters (lm). In each case the number of megakaryocytes was counted manually on 10 images, representing a total area of 8.691 mm2, and converted to megakaryocytes per square millimeter. Morphometric analysis was performed in a blinded fashion without knowledge of JAK2 mutational status.

Clinical Data Complete clinical data were available for 92 patients. Laboratory data included complete blood count, presence or absence of circulating blasts, large or giant platelets, serum ferritin, and lactate dehydrogenase. Clinical parameters included splenic size; events of arterial thrombosis (myocardial infarction, stroke, or transient ischemic attack); venous thrombosis (deep vein, splanchnic vein, retinal vein, or cerebral sinus thrombosis), including pulmonary arterial thromboembolism; and major hemorrhages (gastrointestinal, intracranial, or epistaxis).

Statistical Analysis Data were analyzed using SPSS 16.0 (SPSS Inc, Chicago, Illinois). For comparison of mean values between JAK2 WT and JAK2 V617F Ph MPNs, the normal distribution of data was tested using the Kolmogorov-Smirnov test. According to this, either the t test or the Kruskal-Wallis test was used. Equality of variances was considered in the t test using the Levene test. For comparison of frequencies in cross-tabulations, the v2 test and, if appropriate, the 2-sided Fisher exact test were used. A P value ,.05 was considered to indicate a statistically significant difference. For multivariate analysis, all univariate significant parameters were fitted into a binary logistic regression model using a conditional stepwise forward entry of parameters.

RESULTS A total of 112 BM biopsies obtained from patients with previously untreated Ph MPNs were investigated. Using denaturing high-performance liquid chromatography, JAK2 V617F mutation was detected in 24 of 52 ET cases (46.2%), 36 of 38 PV cases (97.5%), and 14 of 22 PMF cases (63.6%). Megakaryocytic Morphology and JAK2 Mutation Status—Vytrva et al

Table 1.

Cellularity, Iron, and Myelofibrosis (MF)

ET

PMF

WT, No. (%) V617F, No. (%) P Value PV V617F, No. (%) WT, No. (%) V617F, No. (%) P Value

JAK2 Total No. of cases Overall cellularity Normal Increased Erythropoiesis Normal Increased Granulopoiesis Normal Increased Iron Absent Decreased (trace) Present Increased (abundant) MF MF 0 MF I MF II MF III

28

24

36

8

14

2 (5.6) 34 (94.4)

0 8 (100)

0 14 (100)

0 36 (100)

2 (25) 6 (75)

7 (50) 7 (50)

5 (13.9) 31 (86.1)

1 (12.5) 7 (78.5)

0 14 (100)

24 (66.7) 11 (28.9)a 1 (2.8)a 0

0 4 (50) 1 (12.5) 3 (37.5)

0 14 (100) 0 0

36 (100) 0 0 0

0 3 (37.5) 3 (37.5) 2 (25)

n.c.

..99 6 (21.4) 22 (78.6)

5 (20.8) 19 (79.2)

21 (75) 7 (25)

16 (66.7) 8 (33.3)

20 (71.4) 8 (28.6)

7 (29.2) 17 (70.8)

3 17 5 3

2 15 4 3

.55

.38

.005

.36

.99 (10.7) (60.7) (17.9) (10.7)

(8.3) (62.5) (16.7) (12.5)

.01

n.c. 28 (100) 0 0 0

24 (100) 0 0 0

.38 3 6 4 1

(21.4) (42.9) (28.6) (7.1)

Abbreviations: ET, essential thrombocythemia; JAK2, Janus kinase 2; n.c., not computed; PMF, primary myelofibrosis; PV, polycythemia vera; WT, wild type. a In PV patients with detectable iron deposits, either hemorrhages or thromboses were clinically manifest.

Because of the low number of PV JAK2 WT cases in our study cohort, these were excluded from further statistical analysis. Semiquantitative results are summarized in Table 1, morphometric and morphologic data of megakaryocytes in Table 2, and clinical and hematologic data in Table 3.

compared with ET JAK2 V617F. In the peripheral blood of ET JAK2 V617F cases there was a significant difference with respect to higher red blood cell count (P ¼ .01), higher hemoglobin (Hb) concentration (P ¼ .01), and less frequent occurrence of large/giant platelets (P ¼ .02) compared with ET JAK2 WT.

Essential Thrombocythemia

Primary Myelofibrosis

A statistically significant difference between ET JAK2 V617F and ET JAK2 WT was detected in BM biopsies regarding the amount of granulopoiesis, which was more often increased in JAK2 V617F (P ¼ .005). In ET JAK2 WT cases the number of megakaryocytes per square millimeter was significantly higher (P ¼ .02), and these were more polymorphous in terms of cytoplasmic circularity (P ¼ .04)

In PMF, significant differences were observed in the morphology of megakaryocytes in both JAK2 subgroups. In PMF JAK2 V617F, megakaryocytes had a larger cytoplasmic area (P ¼ .04), more regular cytoplasmic circularity (P ¼ .001), a lower percentage of megakaryocytes in clusters (P ¼ .049), a lower percentage of megakaryocytes attached to bone trabeculae (P ¼ .05), and a higher percentage of

Table 2.

Morphometric and Morphologic Data of Megakaryocytes ET

JAK2 Total No. of cases Megakaryocytes per mm2 Size and shape Cytoplasmic major diameter, lm Nuclear to cytoplasmic area ratio Cytoplasmic area, lm2 Cytoplasmic circularity Nuclear circularity Localization and distribution, % Megakaryocytes in clusters Megakaryocytes attached to trabeculae Intertrabecular megakaryocytes Megakaryocytes with ‘‘naked’’ nuclei Megakaryocytes with emperipolesis

WT, Mean (SD)

PMF

V617F, P PV V617F, Mean (SD) Value Mean (SD)

28 24 74.8 (41.2) 53.26 (16.8) 32.9 0.24 611.3 0.78 0.67

(3.17) (0.12) (126.8) (0.04) (0.07)

7.1 (17.0) 2.5 (4.4)

32.8 0.28 622 0.80 0.67

(32.8) (0.28) (122.0) (0.03) (0.05)

4.2 (16.4) 3.8 (5.0)

WT, Mean (SD)

V617F, P Mean (SD) Value

36 8 14 .02a 49.12 (23.8) 106.7 (60.7) 77.2 (30.8)

.14

.94 29.7 (4) .12 0.26 (0.05) .76 531.2 (144.9) .04a 0.83 (0.03) .63 0.7 (0.05)

.12 .22 .04a .001a .52

27.1 0.27 385.4 0.76 0.71

(3) 30 (5.4) (0.08) 0.31 (0.04) (97.1) 520.7 (184.9) (0.03) 0.82 (0.03) (0.05) 0.7 (0.06)

Controls, Mean (SD) 10 2.1 (0.13) 26.6 0.26 438.1 0.87 0.73

(0.6) (0.02) (21.5) (0.03) (0.04)

.52 .35

4.7 (6.7) 3.9 (5.5)

56.3 (35.8) 24.3 (26.8) 21.3 (13.6) 8.6 (13.0)

.049a .05a

0 (0) 0 (0)

78.8 (13.6) 91.4 (13.0) 0.1 (0.22) 0.3 (0.34)

.05a .19

100 (0.0) 0 (0)

.33

0 (0)

97.5 (4.4) 0.3 (3.3)

96.3 (5.0) 0.2 (0.2)

.34 .17

96.1 (5.5) 0.01 (0.1)

0.1 (0.2)

0.1 (0.1)

.06

0 (0)

0 (0)

0.03 (0.08)

Abbreviations: ET, essential thrombocythemia; JAK2, Janus kinase 2; PMF, primary myelofibrosis; PV, polycythemia vera; WT, wild type. a Controls. Arch Pathol Lab Med—Vol 138, September 2014

Megakaryocytic Morphology and JAK2 Mutation Status—Vytrva et al 1205

Table 3.

Clinical and Hematologic Data

ET JAK2 Total No. of 28 cases (male patients/ female patients) Age, y (SD) 58.3 Splenomegaly, No. (%) Yes 6 No 16 Arterial thrombosis, No. (%) Yes 5 No 19 Venous thrombosis, No. (%) Yes 5 No 19 Hemorrhage/ bleeding, No. (%) Yes 1 No 23 Peripheral blood, mean (SD) 4.5 Erythrocytes, 3106/lL Hemoglobin, 1.33 g/dL Mean cell 88.7 volume, fL 10.1 White cells, 3103/lL Neutrophils, 71.0 % 859.2 Platelets, 3103/lL Large or giant platelets, No. (%) Yes 8 No 14 Circulating blasts Yes 4 No 19 LDH, U/L, 309.3 mean (SD) Ferritin, ng/ 113 600 mL, mean (SD)

WT (12/16)

PMF V617F

P Value

24 (12/12)

PV V617F 36 (23/13)

(15.6)

61.2 (11.1)

.15

(27.3) (72.7)

4 (20) 16 (80)

.72

62.4 (13) 11 (39.3) 17 (60.7)

WT

V617F

8 (7/1)

14 (5/9)

66.3 (11.1) 6 (85.7) 1 (14.3)

72.9 (10.9)

P Value

.20 ..99

9 (90) 1 (10) ..99

(20.8) (79.2)

2 (10) 18 (90)

.43

7 (25) 21 (75)

0 (0) 7 (100)

1 (10) 9 (90) n.c.

(20.8) (79.2)

3 (15) 17 (85)

.71

1 (3.6) 27 (96.4)

0 (0) 7 (100)

0 (0) 10 (100) n.c

(4.2) (95.8)

1 (5) 19 (95)

..99

0 (0) 28 (100)

0 (0) 7 (100)

0 (0) 10 (100)

(0.7)

4.9 (0.5)

.01

6.2 (1.1)

3.2 (0.92)

4.1 (1.1)

.09

(0.2)

1.46 (0.1)

.01

1.73 (0.22)

0.89 (0.22)

1.26 (0.29)

.009

(7.1)

82.9 (18.6)

.35a

85.1 (40.4)

88.4 (9.7)

91.1 (7.3)

.54

a

(4.9)

9.5 (2.4)

.45

11.6 (5.7)

6.2 (3.3)

16.9 (10.5)

.01

(10.7)

69.4 (11.1)

.64

74.5 (12.3)

68.0 (12.3)

72.1 (9.5)

.47

(168.5)

762 (153)

.11

529.3 (214.9)

455.4 (231.1)

369.7 (226.6)

.46 n.c.

(36.4) (63.6)

1 (5) 19 (95)

.02

7 (24.1) 22 (75.9)

2 (50) 2 (50)

4 (40) 6 (60) .15

(17.4) (82.6) (106)

2 (9.5) 19 (90.5) 260.4 (78.2)

(113 700) 93 000 (116 600)

.67 .17 .71

2 (6.9) 27 (93.1) 252.1 (74.5)

5 (28.6) 2 (71.4) 695.6 (371)

3 (30) 7 (70) 399.3 (141.1)

45 900 (40 400) 643 500 (262 300) 214 900 (159 700)

.04 .04

Abbreviations: ET, essential thrombocythemia; JAK2, Janus kinase 2; LDH, lactate dehydrogenase; n.c., not computed; PMF, primary myelofibrosis; PV, polycythemia vera; WT, wild type. a Kruskal-Wallis test.

megakaryocytes in intertrabecular spaces (P ¼ .05). In peripheral blood, PMF JAK2 V617F subgroups showed a higher Hb concentration (P ¼ .009), a higher white blood cell count (P ¼ .01), a lower lactate dehydrogenase concentration (P ¼ .04), and a lower serum ferritin level (P ¼ .04) compared with PMF JAK2 WT. In PMF cases the amount of BM iron differed significantly, because all PMF JAK2 V617F cases showed decreased iron stores (P ¼ .01). 1206 Arch Pathol Lab Med—Vol 138, September 2014

Similarities and Differences in JAK2 V617F ET, PV, and PMF Although the morphology of megakaryocytes was quite different in ET JAK2 V617F and PV JAK2 V617F, there were similarities in localization, distribution, and number of these cells (Table 2). Although the morphology of megakaryocytes differed in ET JAK2 V617F and PMF JAK2 V617F cases, there were striking similarities in PV JAK2 V617F and PMF JAK2 Megakaryocytic Morphology and JAK2 Mutation Status—Vytrva et al

Largest-appearing forms of megakaryocytes (largest maximum diameter and largest cytoplasmic area) with polymorphic nuclei (lowest circularity) in essential thrombocythemia (ET) Janus kinase 2 V617F (JAK2 V617F) (A) and ET JAK2 wild type (JAK2 WT) (B) compared with other Philadelphianegative myeloproliferative neoplasms (Ph– MPNs). Morphologic similarities of megakaryocytes (cytoplasmic area and cytoplasmic circularity) in primary myelofibrosis (PMF) JAK2 WT (C) and polycythemia vera JAK2 V617F (D). Distinct morphology of megakaryocytes (smallest cytoplasmic stain, original magnifications 3600). area) in PMF JAK2 WT (E) compared with other Ph– MPNs (May-Grunwald-Giemsa ¨

Arch Pathol Lab Med—Vol 138, September 2014

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V617F cases, especially with regard to morphometric characteristics (Figure). Clinical parameters of ET JAK2 V617F and PMF JAK2 V617F cases revealed higher but not significant values of red blood cells and Hb compared with their respective JAK2 WT counterparts, with the highest values in PV JAK2 V617F. Decreased serum ferritin levels correlated inversely with increased erythropoiesis in ET JAK2 V617F and PMF JAK2 V617F compared with their respective WT counterparts. Serum ferritin level even was lowest in PV JAK2 V617F cases, indicating relatively increased erythropoiesis. Multivariate Analysis In multivariate analysis, platelet count (P , .001) and peripheral white blood cell count (P ¼ .02), as well as Hb concentration (P , .001), were significantly associated with JAK2 V617F mutation. COMMENT JAK2 is a tyrosine kinase–mediating signal transducer and activator of the signaling pathways for the transcription signaling proteins mitogen-activated protein kinase and phosphatidylinositol 3-kinase, leading to proliferation and transformation of hematopoietic progenitors.3–7 Whereas most PV patients show JAK2 V617F, about half of patients with PMF or ET lack this specific mutation.11 We investigated whether Ph MPNs with JAK2 V617F are associated with characteristic clinical parameters and morphologic features in the BM biopsy. We also studied whether similar morphologic or clinical characteristics within JAK2 V617F and JAK2 WT subgroups might imply a pathogenetic relationship among classical Ph– MPN entities. A hallmark of the histopathologic diagnosis of MPNs is the number and appearance of megakaryocytes.2 Our morphometric studies of megakaryocytes in Ph– MPN demonstrated the similarities of these cells in PV JAK2 V617F and PMF JAK2 V617F, suggesting a pathogenetic relationship between the two entities (Figure, C and D). From the morphologic point of view, one could hypothesize that some cases of PMF JAK2 V617F represent an advanced stage of PV JAK2 V617F, comparable or even identical to the ‘‘spent phase’’ of PV JAK2 V617F. In contrast, we found striking morphologic differences in megakaryocytes between PV JAK2 V617F and PMF JAK2 WT (Figure, D and E), as well as both subgroups of ET. In ET, megakaryocytes are the largest of all Ph MPNs (Figure, A and B). In addition, the nuclei are more polymorphic (less circular) and are characterized by a less regular cytoplasmic outline compared with normal forms, which was also observed by Thiele et al.18,19 Our morphologic findings concerning megakaryocytes do not support a close relationship between ET JAK2 V617F and PV JAK2 V617F (Figure, A and D), which is also in agreement with the morphologic data of Gianelli et al.20 In addition to the morphometric differences in megakaryocytes between ET JAK2 V617F and PV JAK2 V617F, these two entities only share some architectural similarities in the BM. Nevertheless, it is important to stress that the morphometric similarities of megakaryocytes are only observed between the PV JAK2 V617F and PMF JAK2 V617F subgroups. Ph MPNs with JAK2 V617F are associated with increased hematopoiesis in BM compared with Ph MPNs with JAK2 WT, which is reflected in BM morphology and complete blood count. We found a significant increase in granulo1208 Arch Pathol Lab Med—Vol 138, September 2014

poiesis in cases of ET JAK2 V617F compared with cases of ET JAK2 WT. The Hb levels and red blood cell counts were higher in ET JAK2 V617F and PMF JAK2 V617F cases. In addition, decreased serum ferritin as a surrogate marker of increased erythropoiesis was observed in ET JAK2 V617F and PMF JAK2 V617F, being statistically significant in the latter. These data are in agreement with the findings of Rudzki et al,21 who reported that the JAK2 V617F mutation skews all Ph MPNs to a more ‘‘erythremic’’ and less ‘‘thrombocythemic’’ phenotype. Targeted therapies for Ph MPNs focus on the inhibition of Jak2 protein activity.13,22,23 To date, clinical trials have enrolled patients with mutated as well as unmutated forms of the gene because Jak2 inhibitors inhibit both mutated and unmutated forms of Jak2 by targeting the ATP-binding site. The clinical trials have shown significant improvements (eg, in splenic size) in both groups of patients. The morphometric study described here could potentially contribute to an understanding of the phenotypic pleiotropy of Ph MPNs by elaborating on clinical-pathologic relationships in the various entities. Our findings could also be helpful in studies exploring the mechanistic effects of JAK2 V617F in hematopoietic cell lines and in assessment of therapies targeting the Jak2 signaling pathway. This work has been supported by a Union for International Cancer Control (UICC) Raisa International Cancer Technology Transfer (ICRETT) Fellowship (Dr Vytrva), which is gratefully acknowledged. We would also like to thank the Bank of Austria for financial support of the Visiting Scientist Program at the Medical University of Graz (Dr Vytrva). At the Institute of Pathology at the Medical University of Graz, Martina Wild, Elisabeth Steinbauer, Mohamed Al-Effah, Josef Bedrac, and Astrid Kainz are acknowledged for excellent laboratory work on BM biopsies, and Andrea Lackner is acknowledged for excellent technical assistance. Dr Bjorkholm would like to thank the Adolf H. Lundin Charitable Foundation. References 1. Tefferi A, Vardiman JW. Classification and diagnosis of myeloproliferative neoplasms: the 2008 World Health Organization criteria and point-of-care diagnostic algorithms. Leukemia. 2008;22(1):14–22. 2. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Hematopoietic and Lymphoid Tissues. Lyon, France: International Agency for Research on Cancer; 2008. 3. James C, Ugo V, Le Couedic JP, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature. 2005; 434(7037):1144–1148. 4. Baxter EJ, Scott LM, Campbell PJ, et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet. 2005;365(9464): 1054–1061. 5. Levine RL, Wadleigh M, Cools J, et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell. 2005;7(4):387–397. 6. Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med. 2005;352(17):1779–1790. 7. Zhao R, Xing S, Li Z, et al. Identification of an acquired JAK2 mutation in polycythemia vera. J Biol Chem. 2005;280(24):22788–22792. 8. Rane SG, Reddy EP. Janus kinases: components of multiple signaling pathways. Oncogene. 2000;19(49):5662–5679. 9. Yamaoka K, Saharinen P, Pesu M, Holt VE III, Silvennoinen O, O’Shea JJ. The Janus kinases (Jaks). Genome Biol. 2004;5(12):253. 10. Kilpivaara O, Levine RL. JAK2 and MPL mutations in myeloproliferative neoplasms: discovery and science. Leukemia. 2008;22(10):1813–1817. 11. Hussein K, Bock O, Kreipe H. Histological and molecular classification of chronic myeloproliferative disorders in the age of JAK2: persistence of old questions despite new answers. Pathobiology. 2007;74(2):72–80. 12. Tefferi A, Gilliland DG. Oncogenes in myeloproliferative disorders. Cell Cycle. 2007;6(5):550–566. 13. Santos FP, Kantarjian HM, Jain N, et al. Phase 2 study of CEP-701, an orally available JAK2 inhibitor, in patients with primary or post-polycythemia vera/ essential thrombocythemia myelofibrosis. Blood. 2010;115(6):1131–1136. 14. Beham-Schmid C, Apfelbeck U, Sill H, et al. Treatment of chronic myelogenous leukemia with the tyrosine kinase inhibitor STI571 results in marked regression of bone marrow fibrosis. Blood. 2002;99(1):381–383.

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15. Bartl R, Frisch B, Burkardt R. Die Knochenmarksbiopsie. Basel, Switzerland: Karger; 1984. 16. Stuart-Smith SE, Hughes DA, Bain BJ. Are routine iron stains on bone marrow trephine biopsy specimens necessary? J Clin Pathol. 2005;58(3):269–272. 17. Rasband W. ImageJ [computer program]. Bethesda, MD: National Institutes of Health; 2008. http://rsbweb.nih.gov/ij/. Accessed January 7, 2013. 18. Thiele J, Funke S, Holgado S, Choritz H, Georgii A. Megakaryopoiesis in chronic myeloproliferative diseases: a morphometric evaluation with special emphasis on primary thrombocythemia. Anal Quant Cytol. 1984;6(3):155–167. 19. Thiele J, Schneider G, Hoeppner B, Wienhold S, Zankovich R, Fischer R. Histomorphometry of bone marrow biopsies in chronic myeloproliferative disorders with associated thrombocytosis–features of significance for the diagnosis of primary (essential) thrombocythaemia. Virchows Arch A Pathol Anat Histopathol. 1988;413(5):407–417.

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20. Gianelli U, Iurlo A, Vener C, et al. The significance of bone marrow biopsy and JAK2V617F mutation in the differential diagnosis between the ‘‘early’’ prepolycythemic phase of polycythemia vera and essential thrombocythemia. Am J Clin Pathol. 2008;130(3):336–342. 21. Rudzki Z, Sacha T, Stoj A, et al. The gain-of-function JAK2 V617F mutation shifts the phenotype of essential thrombocythemia and chronic idiopathic myelofibrosis to more ‘‘erythremic’’ and less ‘‘thrombocythemic’’: a molecular, histologic, and clinical study. Int J Hematol. 2007;86(2):130–136. 22. Koppikar P, Levine RL. JAK2 and MPL mutations in myeloproliferative neoplasms. Acta Hematol. 2008;119(4):218–225. 23. Pesu M, Laurence A, Kishore N, Zwillich SH, Chan G, O’Shea JJ. Therapeutic targeting of Janus kinases. Immunol Rev. 2008;223:132–142.

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Megakaryocytic morphology and clinical parameters in essential thrombocythemia, polycythemia vera, and primary myelofibrosis with and without JAK2 V617F.

Megakaryocytes are the "hallmark" of Philadelphia chromosome-negative myeloproliferative neoplasms, such as essential thrombocythemia, polycythemia ve...
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