Abstract. Hematopathology / Early Prepolycythemic Phase of PV

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1 Hematopathology / Early Prepolycythemic Phase of PV The Significance of Bone Marrow Biopsy and JAK2 V617F Mutation in the Differential Diagnosis Between the Early Prepolycythemic Phase of Polycythemia Vera and Essential Thrombocythemia Umberto Gianelli, MD, 1 Alessandra Iurlo, MD, 2 Claudia Vener, MD, 3 Alessia Moro, MD, 1 Elisa Fermo, 2 Paola Bianchi, 2 Daniela Graziani, 1 Franca Radaelli, MD, 2 Guido Coggi, MD, 1 Silvano Bosari, MD, 1 Giorgio Lambertenghi Deliliers, MD, 3 and Alberto Zanella, MD 2 Key Words: JAK2 V617F mutation; Polycythemia vera; Essential thrombocythemia; Early prepolycythemic polycythemia vera; Bone marrow biopsy; Differential diagnosis Abstract It has been suggested that polycythemia vera (PV) could be preceded by an early phase of the disease (e-pv), in which the increase in the red cell parameters is lower than required for a PV diagnosis. In this study, we compared the clinicopathologic and molecular features of 17 patients with e-pv with those of 14 patients with essential thrombocythemia (ET) and 19 with PV. The results for e-pv were more similar to those for PV than for ET. In fact, patients with e-pv were characterized by an increase in the red cell parameters, splenomegaly (P <.05), and hepatomegaly (P =.038), together with hypercellular bone marrow due to increased erythropoiesis and granulopoiesis, associated with megakaryocytic hyperplasia, with pleomorphic aggregates (P <.001). The frequency of the JAK2 V617F mutation was similar in e-pv (16 cases tested [100%]) and PV (18/19 [95%]) but was significantly lower (7/13 [54%]) in ET (P =.0007). We propose a diagnostic algorithm helpful to distinguish ET from the early prepolycythemic phase of PV. Polycythemia vera (PV) is a chronic myeloproliferative disorder (CMPD) in which, according to the World Health Organization (WHO) classification, 1 2 well-defined stages of the disease are clearly recognizable. The polycythemic phase (pp-pv) is associated with increased red cell mass and characterized by a hypercellular bone marrow (panmyelosis) with prominent erythroid and megakaryocytic proliferation, with pleomorphic aspects. The postpolycythemic myelofibrosis is characterized by progressive cytopenia due to ineffective hematopoiesis, together with bone marrow fibrosis, extramedullary hematopoiesis, and hypersplenism. This latter phase could be complicated in 10% to 15% of the cases by the development of spontaneous or therapy-related acute leukemia. 2,3 Recently, it was suggested that PV could be preceded by an early latent phase of the disease, 4 in which, by definition, the increase in the red cell mass or hemoglobin level is lower than required for a PV diagnosis by the updated diagnostic criteria of the Polycythemia Vera Study Group 5 or by the WHO classification. 1 This hypothesis is supported by previous observations: the development of erythrocytosis in the course of essential thrombocythemia (ET), 6 the identification of masked PV in patients with idiopathic thrombocytosis by endogenous erythroid colony assays, 7 and the observation that thrombocytosis may occur in the early PV stage, mimicking ET. 8 Among the laboratory tests useful for the diagnosis of PV, the determination of erythropoietin (EPO) level and endogenous erythroid colony formation has been included in the WHO classification. Unfortunately, while elevated EPO levels suggest tissue hypoxia, as a reactive cause of erythrocytosis, a normal or slight increase of the EPO level does not permit exclusion of secondary erythrocytosis. 9 Moreover, the value of the endogenous erythroid colony technique for the 336 Am J Clin Pathol 2008;130: Downloaded 336 from

2 Hematopathology / Original Article demonstration of EPO-independent erythroid colony formation is costly, time-consuming, and not generally available. 10 Overexpression of the PRV-1 gene has been reported as a molecular marker for the diagnosis of PV. 11,12 However, the PRV-1 gene is expressed in other CMPDs and even in normal bone marrow cells and secondary erythrocytosis. 13,14 Recently, an acquired activating mutation of the JAK2 tyrosine kinase (JAK2 V617F ) has been detected in the granulocyte DNA of about 95% of patients with PV. The same mutation has been revealed in 50% to 60% of ET and chronic idiopathic myelofibrosis cases and in 1% to 5% of other myeloid disorders. On the contrary, to date, the JAK2 V617F mutation has not been reported in patients with other neoplasia or in healthy people, and, for this reason, it might carry a strong positive predictive value in the diagnosis of CMPDs More recently, the importance of the JAK2 V617F mutation has further strengthened, and a molecular classification of the CMPDs based on this genetic marker has been proposed. 21 This retrospective study aimed to identify the diagnostic criteria useful for a diagnosis of early (prepolycythemic) phase of PV (e-pv). We examined the clinical, morphologic, and molecular features of a cohort of patients at first observation with thrombocytosis, in the range of ET, and with upperlimit or increased hemoglobin levels, but lower than necessary for a PV diagnosis (e-pv) in whom well-characterized pp-pv developed during follow-up. We compared the features with 2 other groups with well-defined criteria for the diagnosis of ET and pp-pv. On the basis of our results, we propose a diagnostic algorithm that could help distinguish ET from a CMPD consistent with e-pv. Materials and Methods Patients The study population included 17 consecutive patients (9 men and 8 women; M/F ratio, 1.1; median age, 53 years; range, years). At diagnosis, the findings included thrombocytosis (platelet count > /µl [ /L]) and upper limit or increased hemoglobin levels ztable 1z, but lower than necessary for a PV diagnosis. Patients were first seen between 1981 and 2001 and followed up for a median of 16 years (range, 5-26 years). During the course of the disease, well-characterized pp-pv developed (median time of evolution, 9 years; range, 2-17 years). The control groups consisted of 19 patients with pp-pv (median follow-up, 5 years; range, 1-18 years) and 14 patients with ET (median follow-up, 3 years; range, 1-8 years), randomly chosen, fulfilling at the time of the first observation the WHO criteria for the diagnosis of CMPDs. 22 All patients were Philadelphia chromosome negative (Ph ). All the patients provided informed consent. ztable 1z Clinical and Hematologic Parameters at Diagnosis in Patients With e-pv and Control Subjects * e-pv (n = 17) pp-pv (n = 19) ET (n = 14) Age (y) 53 (20-69) 63 (38-85) 62 (18-78) M/F (ratio) 9/8 (1.1) 11/8 (1.4) 7/7 (1.0) Hemoglobin concentration (g/dl) Male ( ) 18.6 ( ) 14.1 ( ) Female 15.2 ( ) 16.7 ( ) 13.3 ( ) RBC count ( /L) Male 5.21 ( ) 6.6 ( ) 4.59 ( ) Female 5.06 ( ) 6.94 ( ) 4.61 ( ) Hematocrit value (%) Male 47.3 ( ) 56 ( ) 42.1 ( ) Female 45.5 ( ) 51 ( ) 42.1 ( ) MCV (fl) Male 88.5 ( ) 83.4 ( ) 90.1 ( ) Female 90.1 ( ) 72 ( ) 89.5 ( ) Platelet count ( 10 9 /L) 720 (614-2,000) 609 (600-1,550) 989 (614-1,383) WBC count ( 10 9 /L) 8 ( ) 10.8 ( ) 7.8 (6-11.1) LDH (U/L) 379 ( ) 510 ( ) 435 ( ) LAP (IU/L) 114 (46-238) 156 (14-307) 77 (39-151) Palpable splenomegaly, No. (%) 8 (47) 12 (63) 1 (7) Palpable hepatomegaly, No. (%) 8 (47) 11 (58) 2 (14) e-pv, early phase of polycythemia vera; ET, essential thrombocythemia; LAP, leukocyte alkaline phosphatase; LDH, lactate dehydrogenase; MCV, mean corpuscular volume; pp-pv, polycythemic phase of polycythemia vera. * Data are given as median (range) unless otherwise indicated. The LAP value was evaluated according to the methods of Kaplow. 25 Values for hemoglobin, hematocrit, and RBC count are given in conventional units; conversions to Système International (SI) units are as follows: hemoglobin (g/l), multiply by 10.0; RBC count ( 10 6 /L), multiply by 1.0; and hematocrit (percentage of 1.0), multiply by MCV and platelet and WBC counts are given in SI units; conversions to conventional units are as follows: MCV (µm 3 ), divide by 1.0; platelet count ( 10 3 /µl), divide by 1.0; and WBC count (/µl), divide by Downloaded from Am J Clin Pathol 2008;130:

3 Gianelli et al / Early Prepolycythemic Ph a s e o f PV Morphologic Analysis Formalin-fixed, paraffin-embedded bone marrow biopsy (BMB) specimens, obtained from the posterior superior iliac spine, were available for all patients. Sections were taken from each block for staining with H&E, Giemsa, Gomori silver impregnation, and Pearls to evaluate morphologic features and iron deposits. The specimens were simultaneously examined and discussed by two of us (U.G. and A.M.) experienced in evaluating BMB specimens, without knowledge of clinical data, using a double-headed microscope. Initially discordant cases were further examined until consensus was reached. The following parameters were examined in detail: (1) the overall bone marrow cellularity in relation to patient age; (2) the quantity of erythropoiesis, granulopoiesis, and megakaryopoiesis; and (3) the presence or absence of left-shifting erythropoiesis and granulopoiesis, defined as an increased number of immature precursors. In regard to megakaryocytes we evaluated the presence or the absence of the following: (1) loose clusters, defined as aggregation of 3 or more megakaryocytes not in contact with each other; (2) dense clusters, defined as aggregation of 3 or more megakaryocytes in contact with each other; (3) pleomorphism of the megakaryocytes, defined as the presence of small and giant megakaryocytes grouped together; (4) maturation defects, defined as megakaryocytes with deviation in the nuclear/cytoplasmic ratio; atypical, hyperchromatic nuclei; and dense, eosinophilic cytoplasm; (5) nuclear hyperlobulation, defined as deep lobulation of the nuclei with a staghorn appearance; (6) bulbous nuclei, defined as hypolobulation of the nuclei with chromatin clumping and a cloud-like or balloon-shaped appearance; and (7) naked nuclei. 23,24 These morphologic features were scored semiquantitatively as follows: 0, absence of the evaluated feature; 1+, slight increase, presence of the evaluated feature 1 or 2 times; 2+, moderate increase, presence of the evaluated feature 3 to 4 times; or 3+, marked increase, presence of the evaluated feature more than 4 times. JAK2 V617F Mutation Analysis We performed JAK2 V617F mutation analysis in 10 of 17 e-pv cases at the first observation, before the polycythemic phase of the disease developed. DNA could be obtained from 9 BMB specimens and from 3 bone marrow smears (for 2 patients, both materials were available). Moreover, JAK2 V617F mutation analysis was conducted on purified granulocytes in all pp-pv cases and all but 1 of the ET cases at diagnosis and in all but 1 of the e-pv cases once the polycythemic phase of the disease developed. Briefly, samples were submitted to standard phenol-chloroform technique for genomic DNA purification. To overcome DNA degradation problems and to improve sensitivity of mutation detection, a protocol of fluorescent allele-specific polymerase chain reaction (PCR) described by McClure et al 16 was applied to BMB specimens in the 9 e-pv cases at diagnosis. On DNA obtained from purified granulocytes and from bone marrow smears, the JAK2 V617F mutation was detected by allele-specific PCR according to the protocol of Baxter et al. 17 Mutational status was confirmed by direct sequencing (ABI PRISM 310 Genetic Analyzer, Applied Biosystems, Warrington, England) using the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems). Statistical Analysis The data were evaluated by using the SPSS 11 statistical package (SPSS, Chicago, IL). All of the hypothesis tests were 2-sided, and all other statistical tests were performed at the 5% significance level (P <.05). Demographic and anamnestic data were analyzed descriptively (frequencies and percentages for qualitative variables; means, medians, and ± SD for quantitative variables). The homogeneity of the population was verified by using the χ 2 test for qualitative parameters and 1-way analysis of variance for quantitative data. The Mann-Whitney U test was used to evaluate the qualitative differences between groups. Results Clinical and Hematologic Features The clinical and laboratory characteristics of patients with e-pv and control subjects are reported in Table 1. In particular, palpable splenomegaly (spleen tip, range, 1-8 cm) was more frequently detected in e-pv and pp-pv than in ET (P =.0058); similarly, hepatomegaly (liver tip, range, 1-4 cm) was present in 8 (47%) of 17 e-pv cases and 11 (58%) of 19 pp-pv cases but in only 2 (14%) of 14 ET cases (P =.038). No differences were observed among the groups in cytogenetic features, the occurrence of major or minor thromboembolic or hemorrhagic events, the presence of paresthesias or erythromelalgia, or the number of deaths. Morphologic Analysis The results of the semiquantitative evaluation of the bone marrow features in the different entities are shown in ztable 2z and ztable 3z. The quantitative differences among the 3 groups were tested by using 1-way analysis of variance. We found a statistically significant difference in overall bone marrow cellularity (P <.001) due to variation in the quantity of erythropoiesis (P <.001), granulopoiesis (P <.001), and megakaryopoiesis (P =.01). By focusing on the quality of the megakaryocytes, we found differences in pleomorphism (P <.001) and the presence of maturation defects (P =.03) and nuclear hyperlobulation (P =.03). 338 Am J Clin Pathol 2008;130: Downloaded 338 from

4 Hematopathology / Original Article ztable 2z Analysis of Morphologic Parameters by Score * e-pv pp-pv ET P Cellularity <.001 Erythropoiesis <.001 Granulopoiesis <.001 Megakaryopoiesis Quality of megakaryocytes Loose clusters Dense clusters Maturation defects Nuclear hyperlobulation Bulbous nuclei Naked nuclei e-pv, early phase of polycythemia vera; ET, essential thrombocythemia; pp-pv, polycythemic phase of polycythemia vera. * 0, absence of the evaluated feature; 1+, slight increase, presence of the evaluated feature 1 or 2 times; 2+, moderate increase, presence of the evaluated feature 3 or 4 times; 3+, marked increase, presence of the evaluated feature >4 times. The quantitative differences among the 3 groups were tested by using the 1-way analysis of variance. ztable 3z Analysis of Morphologic Parameters by Absence or Presence of Selected Characteristics * e-pv pp-pv ET Absent Present Absent Present Absent Present P Erythropoiesis, left shifting <.001 Granulopoiesis, left shifting Pleomorphism of megakaryocytes <.001 Reticulin fibers Iron deposits e-pv, early phase of polycythemia vera; ET, essential thrombocythemia; pp-pv, polycythemic phase of polycythemia vera. * The quantitative differences among the 3 groups were tested by using the 1-way analysis of variance. The qualitative differences in the distribution of the morphologic parameters were statistically examined (Mann- Whitney U test) by comparing e-pv with ET and pp-pv. The first comparison revealed that bone marrow cellularity was higher in e-pv than in ET (P <.001) due to increased erythropoiesis (P <.001) with left shifting (P <.001) zimage 1z and to increased granulopoiesis (P =.004) with left shifting (P =.001). Moreover, in e-pv, there was more pleomorphism of megakaryocytes (P <.001), nuclear hyperlobulation (P =.02), maturation defects (P <.001), and bulbous (P <.001) and naked nuclei (P =.01) zimage 2z. We did not find any significant difference when comparing these morphologic parameters in e-pv and pp-pv. JAK2 V617F Mutational Status JAK2 V617F mutational status was first investigated in 10 of 17 patients with e-pv at the first observation. All investigated cases (100%) were mutated. Subsequently, JAK2 V617F mutational status was tested on purified granulocytes in all pp-pv cases and all but 1 of the ET cases at diagnosis and in all but 1 of the e-pv cases once they developed the polycythemic phase of the disease. Overall, the JAK2 V617F mutation was detected in all e-pv cases (16 [100%]; 10 heterozygous and 6 homozygous), in 18 (95%) of 19 pp-pv cases (13 heterozygous and 5 homozygous), and 7 (54%) of 13 ET cases (6 heterozygous and 1 homozygous) (P =.0007). Discussion Our retrospective study documented that e-pv cases are more similar to pp-pv cases than to ET cases. In fact, e-pv cases had higher red cell parameters and more frequent splenohepatomegaly than ET cases. In patients with e-pv, morphologic analysis revealed a mild to moderate hypercellular marrow for the patients age due to increased erythropoiesis and granulopoiesis, both with left shifting (increase of immature precursors). Megakaryocytic hyperplasia, with pleomorphic aggregates Downloaded from Am J Clin Pathol 2008;130:

5 Gianelli et al / Early Prepolycythemic Phase of PV (presence of small to giant megakaryocytes grouped together) and atypical features (nuclear hyperlobulation, maturation defects, and bulbous and naked nuclei), is also a distinctive characteristic. In our series, the JAK2V617F mutation was detectable with a similar frequency in e-pv (100%) and pp-pv (95%) cases, but it was detected significantly less frequently (54%) in ET cases (P =.0007). We investigated JAK2 mutational status in e-pv at first observation, before a well-defined polycythemic phase of the disease developed, in 10 (59%) of 17 patients. In addition, studies concerning the timing of the JAK2 mutation in Ph CMPDs demonstrated the absence of transformation of JAK2 into JAK2+ cases.26 On the basis of our results, we propose a diagnostic algorithm useful for the differential diagnosis between ET and e-pv zfigure 1z. Following this algorithm, all cases of sustained thrombocytosis without identifiable causes should be investigated for the JAK2 mutation. In cases of JAK2V617F+ thrombocytosis, the evaluation of red cell parameters followed by bone marrow examination could allow classification of the cases. In fact, cases with typical e-pv morphologic features (ie, mild to moderate hypercellular marrow with increased erythropoiesis and granulopoiesis, both with left shifting and megakaryocytic hyperplasia, with pleomorphic aggregates and atypical features) could be classified as CMPD consistent with a JAK2V617F+ e-pv. In the same way, cases with typical ET morphologic features (ie, normocellular to slightly hypercellular bone marrow with loose clusters of large and giant megakaryocytes) could be classified as JAK2V617F+ ET. In contrast, cases with a discrepancy between morphologic features and clinical parameters could also be classified as CMPDs, not otherwise classifiable. Similarly, in cases of JAK2V617F thrombocytosis, one should examine the red cell parameters and the bone marrow morphologic features. Cases with increased red cell parameters and e-pv morphologic features could be classified as e-pv likely, and cases with ET morphologic features could be classified as JAK2V617F ET. Follow-up is necessary for patients with discrepancies between morphologic and clinical findings to exclude reactive processes. The proposed algorithm is supported by data reported in the literature. In fact, our clinical and morphologic results agreed with the study of Thiele et al,4 who reported a high frequency of thrombocytosis, with platelet counts exceeding /µL ( /L), in 59% of patients with e-pv and confirmed their description of the typical bone marrow features. Moreover, the detection of the JAK2V617F mutation in all cases of e-pv in our series supports the concept that the most important diagnostic tool for the diagnosis of PV is now represented by JAK2 mutational screening. In fact, about 95% of PV cases were JAK2V617F mutated, and recently, it has been demonstrated that nearly all JAK2V617F PV cases carry 340 Am J Clin Pathol 2008;130: Downloaded 340 from zimage 1z Increased erythropoiesis with left shifting and pleomorphic megakaryocytes are evident in the bone marrow of early polycythemia vera (Giemsa, 20). A B zimage 2z A, Hypercellular bone marrow due to increased erythropoiesis and granulopoiesis, associated with megakaryocytic hyperplasia, and pleomorphic aggregates are typical features of early polycythemia vera (H&E, 20). B, Normocellular bone marrow and increased numbers of large to giant megakaryocytes grouped in loose clusters are typical features of essential thrombocythemia (H&E, 20). different exon 12 JAK2 mutations.27 For this reason, a JAK2 mutation is present in virtually all patients with PV, and it is possible to consider it a diagnostic marker of the disease.28 In addition, the frequency of JAK2V617F in ET and chronic idiopathic myelofibrosis is about 50%, and this fact represents a diagnostic complement to their diagnosis.

6 Hematopathology / Original Article - Sustained platelet count > /L - No other causes of thrombocytosis JAK2 V617F JAK2 V617F + JAK2 V617F Normal range* Hb <18.5 g/dl M Normal range* Hb <16.5 g/dl F Hb within the normal range* Normal range* Hb <18.5 g/dl M Normal range* Hb <16.5 g/dl F Hb within the normal range* BOM BOM BOM BOM e-pv morphology ET morphology e-pv morphology ET morphology e-pv JAK2+ Exclude reactive CMPDs-nos JAK2+ ET JAK2+ e-pv likely ET JAK2 processes zfigure 1z Diagnostic algorithm based on JAK2 mutational status and bone marrow biopsy examination. Values for hemoglobin are given in conventional units; to convert to Système International (SI) units (g/l), multiply by The platelet count is given in SI units; to convert to conventional ( 10 3 /µl), divide by 1.0. * Normal ranges are within the method specific reference range for age, sex, and altitude of residence. BOM, bone marrow; CMPDs, chronic myeloproliferative disorders; e-pv, early phase of polycythemia vera; ET, essential thrombocythemia; Hb, hemoglobin; nos, not otherwise classifiable; pp-pv, polycythemic phase of polycythemia vera. The great importance of JAK2 V617F in the diagnosis and classification of the Ph CMPDs has been universally accepted, and a molecular classification based on the presence or absence of this mutation has been proposed. 21 According to this classification, in the absence of coexisting secondary erythrocytosis, the presence of the JAK2 V617F mutation in a patient with an increased red cell mass is sufficient for the diagnosis of PV. These considerations are reinforced in the very recently published proposals for the revision of the WHO criteria for the diagnosis of Ph CMPDs, 28 in which the increased red cell volume and the JAK2 mutation are considered the 2 major criteria for the diagnosis of PV. Finally, it is important to stress that the clinical characteristics of patients with e-pv do not permit identification by the updated diagnostic criteria of the Polycythemia Vera Study Group 5 or by the WHO classification. 1 Moreover, the possibility of identifying PV at an early stage may be of some clinical value because a higher incidence of thrombotic events has been reported in PV than in ET. 29,30 The results of our study demonstrate that at least in a subset of patients, the polycythemic phase of PV could be preceded by an early (prepolycythemic) phase of the disease, frequently manifesting with thrombocytosis and mimicking ET. This condition could precede the development of pp-pv for several years. A diagnostic algorithm based on JAK2 mutation screening and bone marrow examination could distinguish classical ET from this early phase of PV. Multicenter prospective studies based on larger series of patients are needed to verify the diagnostic usefulness of the proposed algorithm and to define whether e-pv should be considered a well-defined entity and be included in the current classification schemes. From the 1 Pathology Unit, Department of Medicine, Surgery, and Odontology, University of Milan, San Paolo Hospital and Policlinico IRCCS Hospital, Mangiagalli and Regina Elena Foundation, Milan, Italy; 2 Hematology Unit II, Policlinico IRCCS Hospital, Mangiagalli and Regina Elena Foundation, Milan; and 3 Hematology I Bone Marrow Transplant Unit, University of Milan, IRCCS Ospedale Maggiore Policlinico, Mangiagalli and Regina Elena Foundation, Milan. Supported by research grant from the IRCCS Ospedale Maggiore Policlinico, Mangiagalli and Regina Elena Foundation, Milan, Italy. Downloaded from Am J Clin Pathol 2008;130:

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