(2014) 28, 1191–1195 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu

REVIEW Rethinking the diagnostic criteria of vera

T Barbui1, J Thiele2, AM Vannucchi3 and A Tefferi4

The aim of this review is to critically address the validity and clinical applicability of three major diagnostic classification systems for polycythemia vera (PV), that is, those proposed by the Polycythemia Vera Study Group (PVSG), the British Committee for Standards in Haematology (BCSH) and the World Health Organization (WHO). Special focus is on which one of the three red cell parameters (—HB, —HCT and red cell mass—RCM) should be used as the diagnostic hallmark of PV. The revised BCSH employed a persistently raised HCT level as the first diagnostic criterion in combination with the presence of a JAK2V617F mutation. On the other hand, the WHO classification used a raised HB value as a surrogate for increased RCM in association with molecular markers and for the first time, the (BM) morphology was included as a minor criterion. Ongoing controversy and discussion regards the use of certain threshold values for HCT and HB as surrogates for RCM as well as the existence of prodromal- latent disease, so-called masked PV (mPV). It has been shown that mPV can be recognized in patients not meeting the required HB or HCT threshold levels by both the WHO and BCSH criteria. These cases present with the same baseline clinical features as overt PV but present worsened survival. A critical reappraisal of the WHO criteria may suggest either to reduce the thresholds for HB or to consider HCT values as major diagnostic criterion, as in the BCSH, in association with JAK2V617F mutation. The clinical utility of using HCT as reference variable is supported also by results of clinical trials which explicitly recommend to use the HCT threshold for monitoring treatment. In questionable cases as in mPV, BM biopsy examinations should be mandated together with mutation analysis.

Leukemia (2014) 28, 1191–1195; doi:10.1038/leu.2013.380 Keywords: polycythemia vera; diagnostic criteria; masked polycythemia; bone marrow histology; risk factors for survival

INTRODUCTION CLASSIFICATION SYSTEMS The Polycythemia Vera Study Group (PVSG) was the first Polycythemia Vera Study Group to establish consensus diagnostic guidelines in polycythemia As it has been amply emphasized12,14,16,17 the PVSG criteria24,25,26 vera (PV) as an instrument for conducting a number of clinical stipulate an elevation of the RCM as the most important feature trials.1 Since then, those criteria have undergone significant for the diagnosis of PV (Table 1), irrespective of the HCT or HB modifications by two other classification systems2–4 that value. A fierce debate is still going as regards surrogate markers of included the JAK2V617F or other functionally similar mutations RCM that could constitute a reliable indicator of the body’s red cell which were found to be present in virtually all patients with content. As shown by two groups, in both males and females a overtorinapparent(masked)PV.5–11 However, controversies raised HB is not always equal to an absolute erythrocytosis and and an intense debate still persist on which one of the three red conversely, an HB concentration below the required cutoff level cell parameters (that is, hemoglobin (HB) level, hematocrit (HCT) could be associated with an increased RCM.13,16 However, there is value and red cell mass (RCM) measurement) should be used a consensus that males and females presenting with sustained as the most reliable indicator of an increased RCM for the HCT values 460% and 456% respectively can be assumed to diagnosis of PV.12–17 Moreover, a conflict of opinion has have an absolute erythrocytosis and therefore do not require frequently been expressed concerning the usefulness and confirmatory studies like RCM.30,31 Prompted by these findings, validity of bone marrow (BM) examinations in this disorder.12,14 the procedure for RCM determination has been abandoned in While overt disease usually presents no diagnostic challenges, daily clinical practice since it is a cumbersome and costly test that prodromal-early stage18–20 or masked PV (mPV)21 and terminal is also suboptimal in its diagnostic accuracy.32,33 In clinical (so-called) spent phases or post-PV myelofibrosis22 may be practice, clinicians are well aware that this key issue will not be difficult to establish.23 resolved any time soon and for this reason the majority of them Aim of this paper is to highlight some of the problems would recommend individual choice on this crucial matter.3 and pitfalls associated with the three major classification Although BM examinations were not part of the diagnostic criteria systems that comprise the original24–26 and slightly revised27–29 established by the PVSG (Table 1), a number of interesting criteria proposed by the PVSG, the original30 and revised findings were reported by this group.23,34,35 In particular, it was guidelines2 reported by the British Committee for Standards reported that 36 (13%) of the 281 pretreatment biopsies carried in Haematology (BCSH) and the revised consensus classifi- out in overt PV cases demonstrated a BM hematopoietic cellularity cation promoted by the World Health Organization (WHO) of o60% and therefore appeared relatively normal.23 However, in 2008.4 this unusual rate of normal BM features in PV patients requires

1Papa Giovanni XXIII Hospital, Research Foundation, Bergamo, Italy; 2Institute of Pathology, University of Cologne, Cologne, Germany; 3Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy and 4Division of Hematology, Mayo Clinic, Rochester, MN, USA. Correspondence: Dr T Barbui, Scientific Director Research Foundation, Ospedale Papa Giovanni XXIII, Piazza O.M.S., 1, Bergamo (Bg) 24127, Italy. E-mail: [email protected] Received 28 November 2013; accepted 4 December 2013; accepted article preview online 19 Decenber 2013; advance online publication, 17 January 2014 Polycythemia vera T Barbui et al 1192 careful assessment since some morphological criteria employed using these criteria for conducting a number of important clinical for BM examinations were not explicitated. In fact, one should be trials that included the assessment of the value of phlebotomy aware that in the elderly population comprising PV patients and the description of the fatal leukemogenicity associated with (median age about 61 years),36 the subcortical BM spaces are and P32.1,25 It has to be acknowledged that the regularly occupied by fat cells and the sole expansion of PVSG clinical studies have significantly influenced clinical practice hematopoietic tissue towards this area implies hypercellularity.37 in PV for the last several decades. Only the discovery of JAK2V617F A second aspect to be considered, in addition to the expansion of and related mutations as a molecular marker in MPN has the granulo- and erythropoiesis compartment, concerns the undermined the utility of the PVSG criteria. features of the numerically expanded lineages.34,37 The distinct features that characterize the megakaryocyte compartment in PV include changes in megakaryopoiesis with British Committee for Standards in Haematology pleomorphous aspect, that is, small, medium-sized, large and Shortly after the seminal discovery of JAK2V617F and related giant that are either dispersed or loosely mutations ushering new insights for the understanding of the clustered and show no significant abnormalities of differentiation pathophysiology of MPN, the formerly proposed diagnostic PV (no dysplastic features).38–41 These characteristics enable a guidelines by the BCSH30 were amended.2 These modifications differentiation between a reactive state (secondary erythrocytosis- focused on a high HCT level (452% in men and 448% in women) polycythemia) and other entities of chronic myeloproliferative maintained for more than 2 months, as the first diagnostic neoplasia (MPN),23,42 particularly if a proper standardization of criterion in patients harboring the JAK2V617F mutation (Table 2). relevant BM parameters is employed for routine evaluation.43,44 In these cases, the measurement of RCM is not required for Moreover, a rate ranging between 10 and 20% of BM reticulin diagnosis when the other two criteria are both met.48 On the fibrosis has been reported in samples at diagnosis of PV, and is other hand, the problem of mutation-positive patients not predominantly of minor degree.37,40,41,45,46 The corresponding meeting the postulated threshold level of HCT cannot be data provided by the PVSG showed a moderate-to-marked undermined. In an international study on 1545 PV patients of increase of reticulin fibrosis in 11% and a slight one in 25% of which 98% presented with JAK2V617F or related mutations and the patients. It should be mentioned that fibrosis greater than 81% with a subnormal level, the median HCT grade 147 is a relatively rare finding in initial BM biopsies of PV level was 57% in men and 54% in women but ranged from 42 to patients and accounts for only 3–5% of samples.40,45,46 78% and 36 to 76%, respectively.36 It is tempting to assume that Noteworthy is that patients presenting with fibrosis were less patients who do not conform with the HCT threshold values may prone to experience thrombotic complications during their clinical represent cases described formerly as idiopathic erythrocytosis course and conversely more prone to develop post-PV or even occult, latent PV in the pre-JAK2V617F era.27,29 myelofibrosis.46 Furthermore, it is conceivable that some of these cases may Altogether the PVSG has to be credited not only for developing be included in the group of initial/pre-polycythemic PV that the first consensus diagnostic criteria in PV,24,25,26 but also for occasionally present with an elevated count and may

Table 1. Diagnostic criteria for polycythemia vera (PV) according to the original PVSG criteria24–26

Category A Category B

Al Total red cell volume, male X36 ml/kg, female X32 ml/kg B1 Thrombocytosis, platelet count4400 Â 109/1 9 A2 Art.O2 saturation X92% B2 Leukocytosis412 Â 10 /1 (no fever or infection) A3 Splenomegaly B3 Leukocyte score4100 (no fever or infection) B4 Serum B124900 pg/ml or unbound B12 binding capacity42200 pg/ml

Al þ A2 þ A3 or A4 establishes PV Al þ A2 þ any two of category B establishes PV Abbreviation: PVSG, Polycythemia Vera Study Group.

Table 2. Modified diagnostic criteria (2) for polycythemia vera (PV) based on the British Committee for Standards in Haematology (BCSH) guidelines30

JAK2-positive polycythemia vera JAK2-negative polycythemia vera

A1 High hematocrit (40.52 in men, 40.48 in women) or raised red cell A1 Raised red cell mass (425% above predicted) or hematocrit mass (425% above predicted)a 40.60 in men, 40.56 in women A2 Mutation in JAK2 A2 Absence of mutation in JAK2 A3 No cause of secondary erythrocytosis A4 Palpable splenomegaly A5 Presence of an acquired genetic abnormality (excluding BCR-ABL) in the hematopoietic cells B1 Thrombocytosis (platelet count 4450 Â 109/l) B2 Neutrophil leukocytosis (neutrophil count410 Â 109/l in non-smokers; 412.5 Â 109/l in smokers) B3 Radiological evidence of splenomegaly B4 Endogenous erythroid colonies or low serum erythropoietin Diagnosis requires both criteria to be present Diagnosis requires A1 þ A2 þ A3 þ either another A or two B criteria aDual pathology (co-existent secondary erythrocytosis or relative erythrocytosis) may rarely be present in patients with a JAK2-positive myeloproliferative disorder. In this situation, it would be prudent to reduce the hematocrit to the same target as for polycythemia vera.

Leukemia (2014) 1191 – 1195 & 2014 Macmillan Publishers Limited Polycythemia vera T Barbui et al 1193 mimic essential .19,20,49 Although reluctant to Although the WHO recognizes precursor-prodromal stages of include BM morphology into the set of diagnostic features for PV4 that at onset may present with a significant thrombocytosis PV,12 the authors of the BCSH guidelines express a more positive and thus could clinically mimic essential thrombocythemia,19,20,49 attitude by admitting that such an investigation is especially there are still significant uncertainties and debates regarding the useful in patients with erythrocytosis who do not fulfill the PV controversial issue of occult-latent (inapparent) or mPV.21 Early criteria and may help to distinguish between MPN and secondary diagnosis of PV is warranted to prevent fatal thrombotic and reactive causes as well.30,48 This has been confirmed in a complications that may occur a long period before a diagnosis recently published study on a group of 30 patients with HB/HCT based on current criteria may be definitely assessed.57,58 These levels in the low diagnostic ranges because in 28 cases, points have been addressed by two recent studies focusing harboring the JAK2V617F mutation, BM findings were in keeping on mPV.21,58 The occurrence of an mPV was established in with PV.17 140 patients presenting with JAK2V617F positivity and a BM morphology strictly conforming with WHO criteria yet presenting a HB level of o18.5 g/dl in males (range 16.0–18.4) and o16.5 g/dl World Health Organization in females (range 15.0–16.4).21 When comparing this cohort with Prompted by the new molecular findings of BCR-ABL-negative 257 patients presenting with overt PV, male predominance, a MPNs an international panel of clinical investigators, scientists and higher incidence of previous history arterial and hematopathologists with expertise in these disorders proposed thrombocytosis was highlighted. Incidence of recurrent a revision of the 2001 WHO diagnostic criteria50 for PV.3 The thrombosis was similar between the two groups but mPV rationale for these proposals, that were later fully adopted by the displayed significantly higher rates of progression to 2008 WHO diagnostic guidelines for PV,4 was that virtually all myelofibrosis and transformation to acute leukemia and an patients with overt or inapparent PV carry the JAK2V617F or other inferior survival. In multivariable analysis, mPV diagnosis was an similar JAK2 mutations which are always absent in either independent predictor of poor survival along with age 465 years 3,51 secondary or spurious polycythemia. Accordingly, the list of and leukocyte count 410 Â 109/l.21 These data suggest that mPV former criteria50 was replaced by two major parameters: evidence represents a heterogeneous condition that may include a small of elevated HB levels (418.5 g/dl in males and 416.5 g/dl in fraction of true ‘early-stage’ PV clinically mimicking essential females) or any other evidence of increased RCM and a positive thrombocythemia at onset19,20,49 and cases resembling JAK2 mutation status. Additionally, the WHO classification myelofibrosis/post-PV myelofibrosis. This unique variant could required so-called minor criteria that included a corresponding help explain the worse outcome compared with classical, overt PV. BM morphology, Epo levels below the reference range and the Interestingly, a comparison between the WHO3,4 and revised BCSH presence of endogenous erythroid colonies (Table 3). WHO- criteria2 revealed a significant decrease in mPV incidence from 35 confirmed diagnosis of PV requires a combination of major and to 15% in the same cohort of 397 patients.58 Noteworthy is that in minor criteria4 to capture occasional cases of occult PV that may both classification systems, mPV patients showed the same be either negative for a known JAK2 mutation or associated with a baseline features and also a worsening of overall survival in mutation burden that is too low to be detected.3,51 Following the comparison with overt PV. Remarkably, the two classification publication of the WHO-revised criteria for PV, the diagnostic systems allowed to stratify mPV patients in two subgroups accuracy of these guidelines was challenged mainly concerning at different survival according to age and leukocyte counts. on the reliability of the HB level as a surrogate of increased Without these two risk factors mPV patients had the same survival RCM.14,17 Furthermore, concerning the minor criteria (Table 3), as overt PV.58 caution has to be expressed regarding an overreliance on the Epo value that are required to be subnormal for being considered as specific for PV, yet in as many as 20% of patients with overt PV Epo levels may be within the normal range.17,36,51–53 Regarding the CONCLUSION test for endogenous erythroid colonies,54–56 one has to be aware A critical review of the 2008 WHO diagnostic criteria3,4 may that this is time consuming and unfortunately, of limited value, suggest either to reduce the corresponding threshold values for because it is not standardized or generally available. Finally, the HB (Table 3) or to introduce HCT values as in the revised BCSH diagnostic value of a characteristic histological BM pattern in PV is guidelines (Table 2). A rationale for shifting from HB to HCT as the supported by several groups37,39–46 (please refer to the paragraph reference parameters derives also from the clinical practice. In fact, concerning the PVSG criteria).23 In this context very recently the clinicians use explicitly the HCT threshold to monitor the effects of validity of BM morphology for PV diagnosis has been further therapy that, in a recent published clinical trial, was confirmed to confirmed.17 be optimally set at o45%.59 Moreover, the revised response

Table 3. Diagnostic criteria for polycythemia vera (PV) according to the 2008 revised WHO guidelines4

Major criteria Minor criteria

1. Hemoglobin 418.5 g/dl in men, 416.5 g/dl in women or 1. Bone marrow biopsy showing hypercellularity for age with trilineage growth other evidence of increased red cell volumea (panmyelosis) with prominent erythroid, granulocytic and megakaryocytic proliferation 2. Presence of JAK2617V F or other functionally similar 2. Serum erythropoietin level below the reference range for normal mutation such as JAK2 exon 12 mutation 3. Endogenous erythroid colony formation in vitro

Diagnosis requires the presence of both major criteria and one minor criterion or the presence of the first major criterion together with two minor criteria Abbreviation: WHO, World Health Organization. aHemoglobin or hematocrit greater than 99th percentile of method-specific reference range for age, sex, altitude of residence or hemoglobin greater than 17 g/dl in men, 15 g/dl in women if associated with a documented and sustained increase of at least 2 g/dl from an individual’s baseline value that cannot be attributed to correction of iron deficiency, or elevated red cell mass 425% above mean normal predicted value.

& 2014 Macmillan Publishers Limited Leukemia (2014) 1191 – 1195 Polycythemia vera T Barbui et al 1194 criteria for PV included also a ‘durable peripheral count 17 Silver RT, Chow W, Orazi A, Arles SP, Goldsmith SJ. Evaluation of WHO criteria 60 remission, defined as HCT lower than 45% without phlebotomies’. for diagnosis of polycythemia vera: a prospective analysis. Blood 2013; 122: In all patients with sustained erythrocytosis not meeting the 1881–1886. relevant threshold values of HB or HCT respectively, in addition to 18 Thiele J, Kvasnicka HM, Zankovich R, Diehl V. The value of bone marrow histology a JAK2-positive mutation status, a BM biopsy examination is indifferentiating between early stage polycythemia vera and secondary (reactive) absolutely mandated as it reliably and reproducibly supports the . Haematologica 2001; 86: 368–374. diagnosis of PV and may substitute the RCM radioisotopic 19 Thiele J, Kvasnicka HM, Diehl V. Initial (latent) polycythemia vera with thrombo- measurement in cases not meeting the full-blown diagnostic cytosis mimicking essential thrombocythemia. Acta Haematol 2005; 113: 213–219. criteria.61,62 20 Gianelli U, Iurlo A, Vener C, Moro A, Fermo E, Bianchi P 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; 30: 336–342. CONFLICT OF INTEREST 21 Barbui T, Thiele J, Gisslinger H, Finazzi G, Carobbio A, Rumi E et al. The authors declare no conflict of interest. Masked polycythemia vera (mPV): results of an international study. Am J Hematol 2013; e-pub ahead of print 31 August 2013; doi:10.1002/ajh.23585. 22 Mesa RA, Verstovsek S, Cervantes F, Barosi G, Reilly JT, Dupriez B et al. 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